CA2515208A1 - Nell peptide expression systems and bone formation activity of nell peptide - Google Patents

Abstract

The invention generally relates to a bone growth factor, and more particularly to compositions including NELL1, articles of manufacture including NELL1 and methods of using NELL1 to induce bone formation. This invention also provides methods for the expression and purification of NELL1 and NELL2 peptides.

Description

NELL PEPTIDE EXPRESSION SYSTEMS
AND BONE FORMATION ACTIVITY OF NELL PEPTIDE
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY
SPONSORED RESEARCH AND DEVELOPMENT
[0001] This work was supported by NIH/NIDR grant number DE9400 and CRC/NIH grant number RR00865. The Government of the United States of America may have certain rights in this invention.
FIELD OF THE INVENTION

[0002] The invention generally relates to a bone growth factor, and more particularly to compositions including NELL1, articles of manufacture including NELL1 and methods of using NELL1 to induce bone formation. This invention also provides methods for the expression and purification of NELL1 and NELL2 peptides.
BACKGROUND OF THE INVENTION

[0003] Growth factors are substances, such as peptides, which affect the growth and differentiation of defined populations of cells in vivo or in vitro.

[0004] Bone formation occurs during development of long bones (endochondral bone formation) and flat bones (intramembraneous bone formation). Further, bone formation occurs during bone remodeling which occurs continuously in adult life in order to preserve the integrity of the skeleton. Finally, bone formation occurs during bone repair, such as when bone wounds occur in a fracture or surgical situation, for example. While separate bone formation mechanisms are thought to be involved in the embryological development of long and flat bones and repair is thought to involve intramembraneous bone formation.

[0005] Bone formation by either mechanism involves the activity of osteoblasts, which are regulated by growth factors. Osteoblasts are derived from a pool of marrow stromal cells (also known as mesenchymal stem cells; MSC). These cells are present in a variety of tissues and are prevalent in bone marrow stroma.
MSC
are pluripotent and can differentiate into a variety of cell types including osteoblasts, chondrocytes, fibroblasts, myocytes, and adipocytes. Growth factors are thought to impact osteogenic cell proliferation, differentiation and osteoblast mineralization, each of which impacts bone formation.

[0006] Autogenous bone has been used, such to repair bone in patients with craniosynostosis and cleft grafting, for example. Craniosynostosis (CS), the premature closure of cranial sutures, affects 1 in 3,000 infants and therefore is one of the most common human congenital craniofacial deformities. Premature suture closure results in cranial dimorphism, which may need surgical correction.
Premature suture closure in human CS may occur by two possibly distinct processes: calvarial overgrowth and bony fusion. Recently, FGF2 and FGFR1 have been implicated in premature cranial suture fusion via CBFA1-mediated pathways (8). Missense mutation of CBFA1 is linked to cleidocranial dysplasia, manifested as delayed suture closure.

[0007] Autologous bone grafting procedures have been performed utilizing autogenous bone, such as from the iliac crest or calvaria. These donor sites are not without associated morbidity including pain, gait disturbance, thigh paresthesia for iliac crest donor sites, and infection, neurologic deficits, and hematomas for calvarial grafts. Further, donor sites may have limited volume and may contribute to increased surgical time and hospital stay.

[0008] Alloplastic grafting materials have also been utilized, and growth factors have been tested in animal models. For example, bFGF has shown potential for use in bone regeneration and repair. Another family of osteogenic growth factors have been described as bone morphogenic protein (BMP). Specifically, BMP-2 recombinant protein has been demonstrated to regenerate mandibular continuity defects and cleft palate defects with results equal to or better than autogenous particulate bone and marrow. BMPs and other osteogenic factors have been studied for use in clinical applications. However, the cost of using minimally effective dosages of BMP has been a limiting factor in clinical use.

[0009] Spinal fusion is a surgical technique in which one more of the vertebrae of the spine are united together so that motion no longer occurs between them.
Indications include: treatment of a fractured (broken) vertebra, correction of deformity, elimination of pain from motion, treatment of instability, and treatment of some cervical disc herniations. The surgery may involve placement of a bone graft between the vertebrae to obtain a solid union between the vertebrae. The procedure also may involve supplemental treatments including the placement of plates, screws, cages, and recently bone morphogenic protein 2 and 7 to assist in stabilizing and healing the bone graft. Autogenous bone grafting has been the clinically preferred method, and yet has about a 30-50% failure rate.
Autogenous bone grafting is a separate surgery and also carries significant morbidity.

[0010] Therefore, safe, effective and affordable compositions and methods are desired to induce bone formation in bone development, disorders, or bone trauma.
SUMMARY OF THE INVENTION

[0011] This invention may provide methods for the expression and purification of NELL1 and NELL2 peptides. In one embodiment, the method includes NELL
peptides, nucleic acid constructs expressing NELL peptides, and cells expressing NELL peptides which may be useful in producing quantities of NELL peptides. In one embodiment, the nucleic acid constructs expressing NELL peptides may further include nucleic acid sequences encoding signal peptides which may facilitate the protein trafficking and post production modification of the NELL peptides in the host cell. In one embodiment, the signal peptide may facilitate the secretion of the peptide from the host cell. Therefore, this invention is advantageous at least in providing quantities of functional NELL peptides which may be purified for clinical or research use.

[0012] The invention may include compositions and substrates including NELL
peptides. In some embodiments, a composition may include NELL1, and may include additional agents which may effect the application, stability, activity, diffusion and/or concentration of the peptide relative to the application site, for example. In some embodiments, a substrate may include cells and/or NELL1 peptide which may facilitate bone repair in the proximity of the implant.

[0013] The invention may include methods of inducing osteogenic differentiation, osteoblastic mineralization and/or bone formation in a variety of clinical applications.

[0014] This invention is advantageous at least in that NELL peptides may provide a greater effect than known growth factors or may enhance the activity of other growth factors. Therefore, lower doses of each growth factor may be used for clinical applications. This is significant at least in that clinical treatments may be more affordable. Further this invention is advantageous at least in that NELL1 enhances osteogenic difFerentiation, osteoblastic mineralization and bone formation, which may improve the clinical rate and effectiveness of treatment with BMP
alone.

DEFINITIONS

[0015] The terms "polypeptide", "peptide" and "protein" may be used interchangeably herein to refer to a polymer of amino acid residues. The terms may apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

[0016] The terms "NELL1 cDNA" may refer to SEQ ID N0:1, 3 and 5 (Figs. 1, 3 & 5 respectively), and "NELL2 cDNA" may refer to SEQ ID N0:7, 9, 11 and 13 (Figs.
7, 9, 11 & 13).

[0017] A NELL1 peptide is a protein which may be expressed by the NELL1 gene or cDNA and includes SEQ ID NO: 2, 4, and 6 (Figs. 2, 4 & 16, respectively).
The NELL1 peptide may include a NELL1 peptide fragment that retains the ability to induce osteogenic cell differentiation, osteoblast differentiation or bone formation.
A NELL2 peptide is a protein which may be expressed by the NELL2 gene or cDNA
and includes SEQ ID NO: 8, 10, 12 and 14 (Figs. 8, 10, 12 and 14, respectively).
The NELL2 peptide may include NELL2 peptide fragments that retain. similar activity to the full NELL2 peptide sequence.

[0018] The term "antibody" may include various forms of modified or altered antibodies, such as an intact immunoglobulin, an Fv fragment containing only the light and heavy chain variable regions, an Fv fragment linked by a disulfide bond, a Fab or (Fab)'2 fragment containing the variable regions and parts of the constant regions, a single-chain antibody and the like. An antibody may include intact molecules as well as fragments thereof, such as, Fab and F(ab')2~ , and/or single-chain antibodies (e.g. scFv) which may bind an epitopic determinant. An antibody may be of animal (such as mouse or rat) or human origin or may be chimeric or humanized. Antibodies may be polyclonal or monoclonal antibodies ("mAb's"), such as monoclonal antibodies with specificity for a polypeptide encoded by a NELL1 or NELL 2 protein.

[0019] The term "capture agent" may refer to molecules that specifically bind other molecules to form a binding complex such as antibody-antigen, lectin-carbohydrate, nucleic acid-nucleic acid, biotin-avidin, and the like.

[0020] The term "specifically binds" may refer to a biomolecule (e.g., protein, nucleic acid, antibody, etc.), refers to a binding reaction which is determinative of the presence biomolecule in heterogeneous population of molecules (e.g., proteins and other biologics). Thus, under designated conditions (e.g. immunoassay conditions in the case of an antibody or stringent hybridization conditions in the case of a nucleic acid), the specified ligand or antibody may bind to its particular "target" molecule and may not bind in a significant amount to other molecules present in the sample.

[0021] The terms "nucleic acid" or "oligonucleotide" may refer to at least two nucleotides covalently linked together. A nucleic acid of the present invention may be single-stranded or double stranded and may contain phosphodiester bonds, although in some cases, nucleic acid analogs may be included that may have alternate backbones, comprising-, for example, phosphoramide, phosphorothioate, phosphorodithioate, omethylphophoroamidite linkages, and/or peptide nucleic acid backbones and linkages. Analog nucleic acids may have positive backbones and/or non-ribose backbones. Nucleic acids may also include one or more carbocyclic sugars. Modifications of the ribose-phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments, for example.

[0022] The term "specific hybridization" may refer to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions, including conditions under which a probe may hybridize preferentially to its target subsequence, and may hybridize to a lesser extent to other sequences.
BRIEF DESCRIPTION OF THE DRAWINGS

[0023]Fig. 1 is a nucleic acid sequence encoding human NELL1 cDNA (SEQ ID

N0:1 ).

[0024]Fig. 2 is an amino acid sequence encoding human NELL1 (SEQ ID

N0:2).

[0025]Fig. 3 is a nucleic acid sequence encoding rat NELL1 cDNA (SEQ ID

N0:3).

[0026]Fig. 4 is an amino acid sequence encoding rat NELL1 (SEQ
ID N0:4).

[0027] Fig. 5 is a nucleic acid sequence encoding mouse NELL1 cDNA (SEQ ID
N0:5).

[0028] Fig. 6 is an amino acid sequence encoding mouse NELL1 (SEQ ID NO:6).

[0029] Fig. 7 is a nucleic acid sequence encoding human NELL2 cDNA (SEQ ID
N0:7).

[0030] Fig. 8 is an amino acid sequence encoding human NELL2 (SEQ ID
N0:8).

[0031] Fig. 9 is a nucleic acid sequence encoding rat NELL2 cDNA (SEQ ID
N0:9).

[0032] Fig. 10 is an amino acid sequence encoding rat NELL2 (SEQ ID N0:10).

[0033] Fig. 11 is a nucleic acid sequence encoding mouse NELL2 cDNA (SEQ
ID N0:11).

[0034] Fig. 12 is an amino acid sequence encoding mouse NELL2 (SEQ ID
NO:12).

[0035] Fig. 13 is a nucleic acid sequence encoding chicken NELL2 (SEQ ID
N0:13).

[0036] Fig. 14 is an amino acid sequence encoding chicken NELL2 (SEQ ID
N0:14).

[0037] Fig. 15 is a flow diagram of one method of producing functional NELL
peptide.

[0038] Fig. 16 illustrates a signal peptide-NELL1-FLAG nucleic acid construct Underlined amino acid sequences are derived from melittin signal peptide. The bond between Alanine and Proline is a putative cleavage site for secretion by High Five cells. The residues from RTVLGFG---- are derived from the mature protein of rat/human NELL1 protein.

[0039] Fig. 17 illustrates the products of extracellular expression of NELL1-FLAG Fig. 17A is a CBB-stained SDS-PAGE gel of UnoQ-eluate containing purified NELL1 peptide produced from high five cells in serum-free medium (Productivity: ca. 3 mg/L medium); Fig. 17B is a Western blotting using anti-FLAG
antibody. Fig. 17C is a CBB-stained SDS-PAGE gel of UnoQ-eluate containing purified NELL1 peptide produced from COS7 cells in serum-free medium (Productivity: < 0.1 mg/L medium); Fig. 17D is a Western blotting using anti-FLAG
antibody.

[0040] Fig. 18 is a Western blot illustrating the extracellular expression of NELL2-FLAG peptide by insect cells in serum-free medium.

[0041] Fig. 19 is a Western blot illustrating the extracellular expression of and NELL2-FLAG peptides by high five cells in two types of serum free medium (Express Five SFM and ESF921 ).

[0042] Fig. 20 is a bar graph depicting alkaline phosphatase induction in fetal rat calvarial cells exposed to NELL1 peptide (1 ng, 10 ng, 100 ng/ml) and BMP4 (100ng/ml).

[0043] Fig. 21A-D are photomicrographs of osteoblasts treated with NELL1 (A &
B 5ng/ml and C & D 50ng/ml).

[0044] Figs. 22A&B are photomicrographs of NELL1 MC3T3 micronodules forming micronodules in the absence of ascorbic acid; Fig. 22B is stained for alkaline phosphatase.

[0045] Figs. 23A-C are photomicrographs depicting mineralization in A) anti-NELL, B) /3-Gal and C) NELL adenoviral constructs; Figs. 23D & E are bar graphs representing osteocalcin and osteoponin levels in each cell group over time.

[0046] Fig. 24 is a photomicrograph of a NELL1 over expressing transgenic mouse stained to depict mineralization demonstrating calvarial overgrowth.

[0047] Figs. 25A & B are photomicrographs of calvaria stained for mineralization in A) NELL1 over expressing transgenic mouse and B) normal littermate, respectively.

[0048] Fig. 26 is a reverse transcriptase polymerase chain reaction blot depicting NELL1 gene expression in fetal rat calvarial cells treated with A) Cbfa1 or B) control.

[0049] Fig. 27A-C are photographs of skeletal staining of the cranium (top), clavicle (middle) and micro-CT of the cranium of A) wild-type, B) Cbfa1+~-, and C) Cbfa1+~-+ NELL1°"erexp mice, respectively.

[0050] Fig. 28A&B are photographs of microCT treated (right) and control (left) calvarial defects; A) BMP2 treated and B) NELL1 treated.

[0051] Fig. 29 is a photograph of microCT treated NELL1 (right) and BMP (left) calvarial defects.

[0052] Fig. 30A&B are photographs of microCT treated NELL1 (right) and control (left) palatal defects.

[0053] Fig. 31A&B are photomicrographs of TUNEL stained cartilage in A) NELL1°"ereXp and B) wild type mice.

[0054] Fig. 32 is a flow diagram of one method of treating a patient to form bone in a selected location.

[0055] Fig. 33A is a schematic depicting one embodiment of an implant; Fig 33B
is a schematic depicting one embodiment of treating a patient to form bone in a selected location.
DETAILED DESCRIPTION

[0056] The present invention is related to agents and methods for inducing bone formation using NELL1. The present invention also is related to methods for the expression and purification of NELL1 and NELL2 proteins.

[0057] NELL1 was identified by Ting and Watanabe simultaneously. NELL1 is a 810 as peptide, distributed primarily in bone. In adults, NELL2 is expressed at high levels in craniofacial bone, and lower levels in long bone. Its role in osteoblast differentiation, bone formation and regeneration has been examined. NELL2 was identified by Watanabe in 1996, and it is a 816 peptide, distributed in neural cells and brain.

[0058] Human NELL1 gene includes at least 3 Cbfa1 response elements in the promoter region. Cbfa1 specifically binds to these response elements in the promoter. NELL1 expression may be under the control of this transcription factors expressed endogenously at least in preosteoblasts, osteoblasts and hypertrophic chondrocytes in development and in adulthood. Cleidocranial disostosis is a developmental cranial defect thought to be caused at least in part by Cbfa disruption.

[0059] In order to study the function of NELL1 and NELL2 peptides, attempts were made to produce and purify the peptide. Unfortunately, NELL1 and NELL2 peptides were unable to be expressed in a number of expression systems.
Specifically, in E. coli direct and S. cerevisiae expression systems no expression s was detected, in E. coli fused and CHO-dhfr expression systems, very low levels of expression occurred. In the baculovirus system, peptides were expressed.

[0060] It was a surprising discovery of this invention that NELL1 and NELL 2 peptides could be expressed at high levels in insect cells, and that the NELL1 and NELL2 peptides expressed in an insect system were functional forms of the protein.

[0061] COS7 cells can be used to produce NELL1 and NELL2 proteins at low levels, such as about 10 micrograms per litter medium, but require serum-containing medium for the expression. Unfortunately, this medium is not suitable for protein production. As for the signal peptides, NELL1 and NELL2 endogenous signal peptides permit peptide low levels of expression in COS7 cells.

[0062] In one embodiment, the invention includes a method of expressing a functional NELL peptide, such as NELL1 or NELL2 peptide, using an insect cell line.
In one embodiment, the insect cell may be a high five cell, Sf9 and other Sf cells.

[0063] In one embodiment, the method may include providing a nucleic acid sequence encoding a NELL peptide, such as NELL1 or NELL2 peptide. The nucleic acid sequence may be a cDNA or genomic DNA, encoding at least a functional portion of a NELL peptide. For example, the nucleic acid sequence may be selected from the group including, but not limited to human NELL1 (SEQ ID
N0:1 ), rat NELL1 (SEQ ID NO:3), mouse NELL1 (SEQ ID N0:5), or human NELL2 (SEQ ID N0:7), rat NELL2 (SEQ ID N0:9), mouse NELL2 (SEQ ID N0:11), chicken NELL2 (SEQ ID N0:13). The nucleic acid sequence may also include sequences such as those with substantial sequence similarity, such as sequences having at least about 75% sequence similarity with any portion of the sequences listed above.

[0064] Further the nucleic acid may include an expression vector for expressing the nucleic acid sequence encoding a NELL peptide, such as NELL1 or NELL2 peptide. For example, the expression vector may be pIZT/V5-His (Invitrogen), and selective markers may also include blastcidin and neomycin.

[0065] Further, the nucleic acid sequence may also include additional nucleic acids which encode reporter products to monitor levels of gene expression, or encode peptide tags which can be visualized using known methods in the art to monitor levels of peptide expression. Additional sequences may be selected so as to not interfere with the expression of the nucleic acid, or the functionality of the expressed peptide product.

[0066) In one embodiment, the method may include providing a nucleic acid sequence encoding a NELL peptide, such as NELL1 or NELL2 peptide, in frame with a nucleic acid sequence encoding a secretory signal peptide. In one embodiment, the secretory signal peptide may be a secretory signal peptide from a secreted bee protein. For example, the nucleic acid sequence may be selected from the group including, but not limited to a melittin signal sequence, drosphila immunoglobulin-binding protein signal sequence, equine interferon-gamma (eIFN-gamma) signal peptide, snake phospholipase A2 inhibitor signal peptide, human and/or chicken lysozyme signal peptide. For mammalian expression systems, a protrypsin leading sequence may also be used.

[0067] In one embodiment, the method may include transfecting an insect cell line with a nucleic acid construct encoding a NELL peptide; and culturing the insect cell line under conditions that permit expression and/or secretion of the NELL
peptide. For example, the cell line may be transfected transiently or stably with the nucleic acid construct encoding a NELL peptide.

[0068] The method may also include collecting secreted NELL peptides and/or purifying NELL peptides for use. Peptide products may be tested for activity in a variety of functional or expression assays. For example in any assay, if a NELL
peptide has a significant effect over a control substance on a given parameter, the NELL peptides may be said to be functional to effect the measured parameter.

[0069] In one embodiment, the invention may include a nucleic acid construct for expressing a NELL peptide, such as NELL1 and/or NELL2 peptide in an insect cell.
The nucleic acid sequence may be a cDNA or genomic DNA, encoding at least a functional portion of a NELL peptide. For example, the nucleic acid sequence may be selected from the group including, but not limited to human NELL1 (SEQ ID
N0:1 ), rat NELL1 (SEQ ID N0:3), mouse NELL1 (SEQ ID N0:5), or human NELL2 (SEQ ID N0:7), rat NELL2 (SEQ ID N0:9), mouse NELL2 (SEQ ID N0:11), chicken NELL2 (SEQ ID N0:13). The nucleic acid sequence may also include sequences such as those with substantial sequence similarity, such as sequences having at least about 75% sequence similarity with any portion of the sequences listed above.
to [0070] The nucleic acid construct may include a nucleic acid sequence encoding a signal peptide. The nucleic acid may include an expression vector for expressing the nucleic acid sequence encoding a NELL peptide. Further, the nucleic acid sequence may include additional nucleic acids which encode reporter products to monitor levels of gene expression, or encode peptide tags which can be visualized using known methods in the art to monitor levels of peptide expression.

[0071] Nucleic acid constructs may comprise expression and cloning vectors should containing a selection gene, also termed a selectable marker, such as a gene that encodes a protein necessary for the survival or growth of a host cell transformed with the vector. The presence of this gene ensures that any host cell which deletes the vector will not obtain an advantage in growth or reproduction over transformed hosts. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate or tetracycline, (b) complement auxotrophic deficiencies.

[0072] Nucleic acid constructs may also include a promoter which is recognized by the host organism and is operably linked to the NELL encoding nucleic acid.
Promoters are untranslated sequences located upstream from the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of nucleic acid under their control, including inducible and constitutive promoters. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, e.g. the presence or absence of a nutrient or a change in temperature. At this time a large number of promoters recognized by a variety of potential host cells are well known.

[0073] A nucleic acid may be operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein which participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.

[0074] In one embodiment, the invention may include cells that express functional NELL peptides. In one embodiment, the cell may be an insect cell.
In one embodiment, the insect cell may be a high five cell.

[0075] In one embodiment, the cell may be transfected with a nucleic acid construct encoding a NELL peptide. For example, the cell line may be transfected transiently or stably with the nucleic acid construct encoding a NELL peptide.
In one embodiment, NELL expressing nucleic acids (e.g., cDNA(s) may be cloned into gene expression vector or viral particles that are competent to transfect cells (such as insect cells).

[0076] The nucleic acid sequence may also include a nucleic acid sequence encoding a NELL peptide, such as NELL1 or NELL2 peptide, in frame with a nucleic acid sequence encoding an insect secretory signal peptide.

[0077] In one embodiment, the invention may include cells that express functional NELL peptides, and may secrete functional proteins.

[0078] In one embodiment, the invention may include a polypeptide (amino acid sequence) comprising a NELL peptide, such as NELL1 or NELL2 peptide, and may include secretory signal peptide.

[0079] For example, the amino acid sequence of the NELL peptide may be selected from the group including, but not limited to human NELL1 (SEQ ID
NO:2), rat NELL1 (SEQ ID N0:4), mouse NELL1 (SEQ ID N0:6), or human NELL2 (SEQ
ID N0:8), rat NELL2 (SEQ ID N0:10), mouse NELL2 (SEQ ID N0:12), chicken NELL2 (SEQ ID N0:14). The amino acid sequence may also include sequences such as those with substantial similarity, such as sequences having at least about 75% sequence similarity with any portion of the sequences listed above, or contain similar active binding domains as NELL1 peptides.

[0080] In one embodimei-it, the invention includes a method purifying NELL1 and/or NELL2 peptides secreted into culture media, according to standard peptide purification protocols, including, but not limited to those described below.

[0081] In one embodiment, whether a selected cell expresses a selected nucleic acid sequence to express and/or secrete a NELL peptide may be examined. In one embodiment, the presence, amount or and/or activity of NELL peptides may be examined.

[0082] In on embodiment, NELL peptides detected and quantified by any of a number of methods well known to those of skill in the art. These may include analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, or various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, western blotting, and the like.

[0083] In one embodiment, Western blot (immunoblot) analysis may be used to detect and quantify the presence of NELL peptides) in a selected sample. This technique may include separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a'suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with the antibodies that specifically bind a target peptide.

[0084] The assays of this invention may be scored (as positive or negative or quantity of target polypeptide) according to standard methods well known to those of skill. in the art. The particular method of scoring may depend on the assay format and choice of label. For example, a Western Blot assay may be scored by visualizing the colored product produced by an enzymatic label. A clearly visible colored band or spot at the correct molecular weight may be scored as a positive result, while the absence of a clearly visible spot or band may be scored as a negative. The intensity of the band or spot may provide a quantitative measure of target polypeptide concentration.

[0085] The NELL1 proteins generated in such expression systems can be used in a manner analogous to the use of bone morphogenic proteins (e.g. BMP-1 through BMP-24). Thus, the NELL1 polypeptide(s) can be used to speed repair of bone fractures or to induce bone repair or replacement under circumstances where natural healing is limited or nonexistent. In addition, the NELL1 polypeptides can be incorporated into bone graft materials. These graft materials can be used in the treatment of fractures or to facilitate the replacement/healing of prostheses or bone transplants and spinal fusion.

[0086] The present invention may also include agents and methods for increasing the degree and/or rate of bone formation. More specifically, the invention may include the systemic and/or local application of agents for increasing bone formation. Clinical indices of a method or agents ability to increase the degree and/or rate of bone formation is evidenced by improvements in bone density at the desired site of bone formation as assessed by DEXA scanning.
Enhanced bone formation in a healing fracture is routinely assessed by regular X-ray of the fracture site at selected time intervals. More advanced techniques for determining the above indices such as quantitative CT scanning may be used.

[0087] In one embodiment, the invention may include, a method of increasing osteogenic cell differentiation comprising increasing the concentration of a gene product in an osteogenic cell, optionally applying a second agent; and inducing the expression of cellular marker of osteoblastic differentiation.

[0088] The method may include increasing the concentration of a NELL1 gene product by applying a NELL1 peptide to an osteogenic cell, and the NELL1 peptide may be selected from the group comprising: SEQ ID N0:2, SEQ ID NO: 4, or SEQ
ID N0:6, or any portion of the NELL peptide which s effective in increasing osteoblastic differentiation. The method may include increasing the concentration of a NELL1 gene product by inducing the expression of an endogenous NELL1 gene, such as by increasing cellular levels of the expression regulating molecule, Cbfa1. The method may include increasing the concentration of a NELL1 gene product by transfecting the osteogenic cell with a nucleic acid construct encoding a NELL1 peptide, and the nucleic acid construct encoding a NELL1 peptide may be selected from the group comprising SEQ ID NO:1, SEQ ID NO: 3, or SEQ ID N0:5.

[0089] Osteogenic cells may include, but are not limited to osteoblasts, mesenchymal cells, fibroblasts, fetal embryonic cells, stem cells, bone marrow cells, dural cells, chondrocytes, chondroblasts and adipose stem cells.

(0090] Osteogenic cells may also include cells that are located within, are in contact with, or migrate towards (i.e., "home to"), bone tissue and which cells directly or indirectly stimulate the formation of bone tissue. As such, the osteogenic cells may be cells that ultimately differentiate into mature osteoblasts cells themselves, i.e., cells that "directly" form bone tissue.

[0091] A second agent may include, but is not limited to: TGF,l3, BMP2, BMP4, BMP7, bFGF, collagen. The second agent may be selected to have a complimentary or synergistic effect with NELL1 in inducing osteoblastic differentiation.

[0092] Cellular markers of osteoblastic differentiation include, but are not limited to increased levels of alkaline phosphatase activity, osteocalcin and osteoponin mRNA expression, BMP7 expression, decorin expression and laminin B1 expression. However, any cellular marker whose activity or expression changes as a result of osteoblastic differentiation may be used as a marker of such.

[0093] In one embodiment, the method of increasing osteoblastic mineralization may include increasing the concentration of a NELL1 gene product in an osteoblastic cell, optionally applying a second agent; and inducing the expression of cellular marker of mineralization.

[0094] The method may include increasing the concentration of a NELL1 gene product by applying a NELL1 peptide to an osteogenic cell, and the NELL1 peptide may be selected from the group comprising: SEQ ID N0:2, SEQ ID NO: 4, or SEQ
ID N0:6, or any portion of the NELL peptide which is effective in increasing osteoblastic mineralization. The second agent may be selected to have a complimentary or synergistic effect with NELL1 in inducing osteoblastic mineralization.

[0095] Cellular markers of osteoblastic mineralization include, but are not limited to increased levels of calcium incorporation. However, any cellular marker whose activity or expression changes as a result of osteoblastic mineralization may be used as a marker of such.

[0096] In one embodiment, a method of increasing intramembraneous bone formation may include increasing the concentration of a NELL1 gene product in a location where bone formation is desired, optionally applying a second agent to approximately the same location region where bone formation is desired; and inducing the formation of intramembraneous bone formation.

[0097] The method may include increasing the concentration of a NELL1 gene product by applying a NELL1 peptide to the location where bone formation is desired, and the NELL1 peptide may be selected from the group comprising: SEQ
ID NO:2, SEQ ID NO: 4, or SEQ ID N0:6, or any portion of the NELL peptide which is effective in increasing intramembraneous bone formation.
is [0098] The second agent may include, but is not limited to TGF,l3, BMP2, BMP4, BMP7, bFGF, collagen, osteogenic cells, bone, bone matrix, tendon matrix, ligament matrix. The second agent may be selected to have a complimentary or synergistic effect with NELL1 in inducing intramembraneous bone formation.

[0099] The formation of intramembraneous bone may be evaluated by microscopic inspection for histology, DEXA scanning, X-ray or CT scanning of bone density in the area where bone formation is desired.

[00100] In one embodiment, a method of increasing endochondral bone formation may include increasing the concentration of a NELL1 gene product in a region where bone formation is desired; optionally applying a second agent to the region where bone formation is desired and at least inducing hypertrophy of chondroblast in the region where bone formation is desired.

[00101] The method may include increasing the concentration of a NELL1 gene product by applying a NELL1 peptide to the location where bone formation is desired, and the NELL1 peptide may be selected from the group comprising: SEQ
ID N0:2, SEQ ID NO: 4, or SEQ ID N0:6, or any portion of the NELL peptide which is effective in increasing endochondral bone formation.

[00102] The second agent may include, but is not limited to TGF /3, BMP2, BMP4, BMP7, bFGF, collagen, osteogenic cells, bone, bone matrix, tendon matrix, ligament matrix. The second agent may be selected to have a complimentary or synergistic effect with NELL1 in inducing endochondral bone formation.

[00103] The formation of endochondral bone may be evaluated by chondroblast hypertrophy as viewed by an increase in hypertrophic and apoptotic chondroblasts, elucidated by TUNEL staining.

[00104] In one embodiment, the invention may include a method of incorporating NELL1 in carriers or substrates, and the resulting substrates.

[00105] In one embodiment, a composition for inducing bone formation may include an effective amount of a first agent to induce bone formation selected from the group including but not limited to a NELL1 peptide, and an agent that alters expression of NELL1 peptide, or an agent that alters the activity of a NELL1 peptide; and optionally a carrier.

[00106] The composition may include a NELL1 peptide selected from the group comprising: SEQ ID N0:2, SEQ ID NO: 4, or SEQ ID N0:6, or any fragment which is effective in inducing bone formation.

(00107] The composition may include a second agent including, but not limited to TGF,l3, BMP2, BMP4, BMP7, bFGF, collagen, bone, bone matrix, tendon matrix or ligament matrix, osteogenic and/or osteoblastic cells.

[00108] In one embodiment, the carrier may be biodegradable, such as degradable by enzymatic or hydrolytic mechanisms. Examples of carriers include, but are not limited to synthetic absorbable polymers such as such as but not limited to poly(a-hydroxy acids) such as poly (L-lactide) (PLLA), poly (D, L-lactide) (PDLLA), polyglycolide (PGA), poly (lactide-co-glycolide (PLGA), poly (-caprolactone), poly (trimethylene carbonate), poly (p-dioxanone), poly (-caprolactone-co-glycolide), poly (glycolide-co-trimethylene carbonate) poly (D, L-lactide-co-trimethylene carbonate), polyarylates, polyhydroxybutyrate (PHB), polyanhydrides, poly (anhydride-co-imide), propylene-co-fumarates, polylactones, polyesters, polycarbonates, polyanionic polymers, polyanhydrides, polyester-amides, poly(amino-acids), homopolypeptides, poly(phosphazenes), poly (glaxanone), polysaccharides, and poly(orthoesters), polyglactin, polyglactic acid, polyaldonic acid, polyacrylic acids, polyalkanoates; copolymers and admixtures thereof, and any derivatives and modifications. See for example, U. S. Patent 4,563,489, and PCT Int. Appl. # WO/03024316, herein incorporated by reference.
Other examples of carriers include cellulosic polymers such as, but not limited to alkylcellulose, hydroxyalkylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, carboxymethylcellulose, and their cationic salts. Other examples of carriers include synthetic and natural bioceramics such as, but not limited to calcium carbonates, calcium phosphates, apatites, bioactive glass materials, and coral-derived apatites.
See for example U.S. Patent Application 2002187104; PCT Int. Appl. WO/9731661;
and PCT Int. Appl. WO/0071083, herein incorporated by reference.

[00109] In one embodiment, the carrier may further be coated by compositions, including bioglass and or apatites derived from sol-gel techniques, or from immersion techniques such as, but not limited to simulated body fluids with calcium and phosphate concentrations ranging from about 1.5 to 7-fold the natural serum concentration and adjusted by various means to solutions with pH range of about 2.8-7.8 at temperature from about 15-65 degrees C. See, for example, U.S.
Patents 6,426,114 and 6,013,591; and PCT Int. Appl. WO/9117965 herein incorporated by reference.

[00110] Other examples of carriers include, collagen (e.g. Collastat, Helistat collagen sponges), hyaluronan, fibrin, chitosan, alginate, and gelatin. See for example, PCT Int. Appls. WO/9505846; WO/02085422, herein incorporated by reference.

[00111] In one embodiment, the carrier may include heparin-binding agents;
including but not limited to heparin-like polymers e.g. dextran sulfate, chondroitin sulfate, heparin sulfate, fucan, alginate, or their derivatives; and peptide fragments with amino acid modifications to increase heparin affinity. See for example, Journal of Biological Chemistry (2003), 278(44), p. 43229-43235, herein incorporated by reference.

[00112] In one embodiment, the substrate may be in the form of a liquid, solid or gel.

[00113] In one embodiment, the substrate may include a carrier that is in the form of a flowable gel. The gel may be selected so as to be injectable, such as via a syringe at the site where bone formation is desired. The gel may be a chemical gel which may be a chemical gel formed by primary bonds, and controlled by pH, ionic groups, and/or solvent concentration. The gel may also be a physical gel which may be formed by secondary bonds and controlled by temperature and viscosity.
Examples of gels include, but are not limited to, pluronics, gelatin, hyaluronan, collagen, polylactide-polyethylene glycol solutions and conjugates, chitosan, citosan & b-glycerophosphate (BST-gel), alginates, agarose, hydroxypropyl cellulose, methyl cellulose, polyethylene oxide, polylactides/glycolides in N-methyl-2-pyrrolidone. See for example, Anatomical Record (2001 ), 263(4), 342-349, herein incorporated by reference.

[00114] In one embodiment, the carrier may be photopolymerizable, such as by electromagnetic radiation with wavelength of at least about 250 nm. Example of photopolymerizable polymers include polyethylene (PEG) acrylate derivatives, PEG
methacrylate derivatives, propylene fumarate-co-ethylene glycol, polyvinyl alcohol is derivatives, PEG-co-poly(-hydroxy acid) diacrylate macromers, and modified polysaccharides such as hyaluronic acid derivatives and dextran methacrylate.
See for example, U.S. Patent 5,410,016, herein incorporated by reference.

[00115] In one embodiment, the substrate may include a carrier that is temperature sensitive. Examples include carriers made from N-isopropylacrylamide (NiPAM), or modified NiPAM with lowered lower critical solution temperature (LCST) and enhanced peptide (e.g. NELL1 ) binding by incorporation of ethyl methacrylate and N-acryloxysuccinimide; or alkyl methacrylates such as butylmethacrylate, hexylmethacrylate and dodecylmethacrylate. PCT Int. Appl. WO/2001070288; U.S. Patent 5,124,151 herein incorporated by reference.

[00116] In one embodiment, where the carrier may have a surface that is decorated and/or immobilized with cell adhesion molecules, adhesion peptides, and adhesion peptide analogs which may promote cell-matrix attachment via receptor mediated mechanisms, and/or molecular moieties which may promote adhesion via non-receptor mediated mechanisms binding such as, but not limited to polycationic polyamino-acid-peptides (e.g. poly-lysine), polyanionic polyamino-acid-peptides, Mefp-class adhesive molecules and other DOPA-rich peptides (e.g. poly-lysine-DOPA), polysaccharides, and proteoglycans. See for example, PCT Int. Appl.
WO/2004005421; WO/2003008376; WO/9734016, herein incorporated by reference.

[00117] In one embodiment, the carrier may include comprised of sequestering agents such as, but not limited to, collagen, gelatin, hyaluronic acid, alginate, polyethylene glycol), alkylcellulose (including hydroxyalkylcellulose), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, blood, fibrin, polyoxyethylene oxide, calcium sulfate hemihydrate, apatites, carboxyvinyl polymer, and polyvinyl alcohol). See for example, United States Patent 6,620,406, herein incorporated by reference.

[00118] In one embodiment, the carrier may include surtactants to promote NELL1 stability and/or distribution within the carrier materials such as, but not limited to polyoxyester (e.g. polysorbate 80, polysorbate 20 or Pluronic F-68).

[00119] In one embodiment, the carrier may include bufFering agents such as, but not limited to glycine, glutamic acid hydrochloride, sodium chloride, guanidine, heparin, glutamic acid hydrochloride, acetic acid, succinic acid, polysorbate, dextran sulfate, sucrose, and amino acids. See for example, U.S. Patent 5,385,887, herein incorporated by reference. In one embodiment, the carrier may include a combination of materials such as those listed above.

[00120] By way of example, the carrier may a be PLGA/collagen carrier membrane. The membrane may be soaked in a solution including NELL1 peptide.

[00121] In one embodiment, an implant for use in the human body may include a substrate including NELL1 in an amount sufficient to induce bone formation proximate to the implant.

[00122] In one embodiment, an implant for use in the human body may include a substrate having a surface including NELL1 in an amount sufficient to induce bone formation proximate to the implant.

[00123] In one embodiment, an implant for use in the human body may include a substrate having a surface including osteogenic cells, and NELL1 in an amount sufficient to induce bone formation. In one embodiment, the implant may be seeded with cells, including but not limited to autologous cells, osteogenic or osteoblastic cells, cells expressing NELL1 or another osteogenic molecule.

[00124] An implant may include a substrate formed into the shape of a mesh, pin, screw, plate, or prosthetic joint. By way of example, a substrate may be in a form of a dental or orthopedic implant, and NELL1 may be used to enhance integration in bone in proximity to the implant. An implant may include a substrate that is resorbable, such as a substrate including collagen.

[00125] In one example, a composition according to this invention may be contained within a time release tablet.

[00126] The NELL1 peptide may be combined with a acceptable carrier to form a pharmacological composition. Acceptable carriers can contain a physiologically acceptable compound that acts, for example, to stabilize the composition or to increase or decrease the absorption of the agent. Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the anti-mitotic agents, or excipients or other stabilizers and/or buffers.

[00127] Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. One skilled in the art would appreciate that the choice of a carrier, including a physiologically acceptable compound depends, for example, on the route of administration.

[00128] The compositions can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable may include powder, tablets, pills, capsules.

[00129] The compositions of this invention may comprise a solution of the peptide dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier for water-soluble peptides. A variety of carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.

[00130] The concentration of NELL1 peptide in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs.

[00131] The dosage regimen will be determined by the clinical indication being addressed, as well as by various patient variables (e.g. weight, age, sex) and clinical presentation (e.g. extent of injury, site of injury, etc.).

[00132] However, a therapeutically effective dose of a NELL1 peptide or agent useful in this invention is one which has a positive clinical effect on a patient or desired effect in cells as measured by the ability of the agent to enhance osteoblastic differentiation, mineralization, bone formation, as described above.

The therapeutically effective dose of each peptide or agent can be modulated to achieve the desired clinical effect, while minimizing negative side effects.
The dosage of the peptide or agent may be selected for an individual patient depending upon the route of administration, severity of the disease, age and weight of the patient, other medications the patient is taking and other factors normally considered by an attending physician, when determining an individual regimen and dose level appropriate for a particular patient.

[00133] Dosage Form. The therapeutically effective dose of an agent included in the dosage form may be selected by considering the type of agent selected and the route of administration. The dosage form may include a agent in combination with other inert ingredients, including adjutants and pharmaceutically acceptable carriers for the facilitation of dosage to the patient, as is known to those skilled in the pharmaceutical arts.

[00134] In one embodiment, the invention may include a method of treating a patient to induce bone formation, comprising administering NELL1 peptide at a therapeutically effective dose in an effective dosage form at a selected interval to enhance bone formation. The method of may further comprise administering at least one secondary agent in the region where bone formation is desired, including but not limited to TGF- ~3, BMP2, BMP4, BMP7, bFGF, collagen, bone, bone matrix, tendon matrix or ligament matrix, osteogenic or osteoblastic cells.

[00135] In one embodiment, a method of treating a patient to induce bone formation may include harvesting mammalian osteogenic cells, increasing the concentration of expression of NELL1 peptide in contact with the osteogenic cells and administering the osteogenic cells to a region where bone formation is desired.

[00136] In one embodiment, bone formation to repair to cranial trauma or cranial defects may be desired, such as occurs in fetuses, infants or adults having cleidocranial disostosis, or cleft palate. In one embodiment, bone formation may be desired in a region of a non-healing bone defect (also known as critical size defect where bone fails to regenerate/heal in the defect). Critical size defect models are studied as a stringent test on agent effecting all bone healing, including long bone fracture, since all bone wound healing is believed to be by membranous (also called intramembraneous) bone formation. For example, long bone fracture and calvarial defect both heal by membranous bone formation. In one embodiment, bone formation may be desired in alveolar bone grafts or alveolar ridge augmentation, or periodontal bone defect. In one embodiment, bone formation may be desired to enhance the integration of implants such as joint or dental implants, or cosmetic surgery onplants.

[00137] In one embodiment, bone formation may be used in alternative or in addition to autologous, autogenous or alloplastic materials for bone grafts.
EXAMPLES

(00138] The following examples are offered to illustrate, but not to limit the claimed invention.

[00139] Example 1. Expression of NELL peptides.

[00140] A cDNA fragment was ligated into the expression vector PiZT/V5-His (3.4kb) (EcoRV site, Invitrogen) and included a melittin signal peptide, BamHl-EcoRl cDNA fragment of the mature rat NELL1 and a FLAG tag sequence. Fig.16 is a depiction of the nucleic acid sequence of the cDNA construct used in this example, and corresponding predicted peptide sequence.

[00141] The High five cells (BTI-TN-5B1-4) were adapted to serum-free medium, and cells were transfected with the NELL1 peptide expression vector. Cells were treated with zeocin so as to select only cell populations expressing the NELL1 FLAG constructs. Surviving cell populations were confirmed to be stable transformants. Extracellular media was collected and tested for the presence of NELL1 peptide. NELL1 peptide was purified and used in functional assays described below.

[00142] Fig. 17A is an illustration of a CBB-stained SDS-PAGE gel of UnoQ-eluate containing purified NELL1 peptide. The medium was applied onto UnoQ
column (Bio-Rad) as described herein. Fig. 4B is an illustration of a Western blot using anti-FLAG antibody depicting NELL1-FLAG expression in reference to a protein ladder. Peptide: 140 kDa (intracellular precursor), 130 kDa (mature form;
90 kDa peptide), 400 kDa (secreted form, homotrimer). In the example above, the productivity of the expression system was about 3 mg NELL1 peptide/L medium.

[00143] Relative to other expression systems which did not express or secrete peptide at all (such as bacterial expression, including yeast) or whose peptide production was extremely low (e.g., E. coli fused peptide system, CHO-dhfr cells, >10mcg/L) production with the systems described (mammalian and insect cells) was surprisingly and substantially more effective at producing large amounts of functional protein.

[00144] Expression and Purification of Recombinant Rat NELL1 Protein. For production of the C-terminally FLAG-tagged NELL1 peptide by insect cells. A
pIZT-NELL1-FLC plasmid was constructed by inserting the rat NELL1 cDNA fused to a FLAG epitope sequence derived from the pTB701-NELL1-FLC plasmid (Kuroda, BBRC) into insect expression vector pIZT/V5-His (Invitrogen). Furthermore, original secretory signal sequence was replaced to honeybee mellitin signal sequence using PCR methods. High Five cells were purchased from Invitrogen, and were cultured in High Five Serum-Free Medium (Invitrogen). High Five cells were transfected with the pIZT-NELL1-FLC plasmid using FuGene6 (Roche).
Forty-eight hours after transfection, cells were selected with 400 mg/ml of Zeocin (Invitrogen). Replace selective medium every 3 to 4 days until the stable expression cell line was established. NELL1 secretion was confirmed using immunoprecipitation and Western blot analyses. High five cells were found to express NELL1 peptides (140-kDa) in the culture medium.

[00145] The recombinant rat NELL1-FLC peptide was purified from the culture medium of Zeocin-resistant High Five cells by anion exchange chromatography using a UNO Q-1 column (Bio-Rad). NELL1 peptide was eluted at 500 mM NaCI.

[00146] For production of the C-terminally FLAG-tagged NELL1 peptide by COS7 cells, a pcDNA3.1-NELL1-FLC plasmid was constructed by inserting the rat NELL1 cDNA linked to a FLAG epitope sequence derived from the pTB701-NELL1-FLC
plasmid into mammalian expression vector pcDNA3.1 (Invitrogen). COS7 cells were cultured in DMEM supplemented with 10% FBS. COS7 cells were transfected with the pcDNA3.1-NELL1-FLC using the endogenous NELL signal peptide plasmid and using electroporation method. Forty-eight hours after transfection, culture medium was subjected to immunoprecipitation and Western blot analyses for NELL1 peptide.

[00147] Fig. 17C is an illustration of a CBB-stained SDS-PAGE gel of UnoQ-eluate. including NELL1-FLAG. These expression studies showed that COS cells did not express functional NELL peptide, without modifying the N terminal of the NELL to increase secretion efficiency such as including a signal sequence.
Fig.

17D is an illustration of a Western blot using anti-FLAG antibody depicting FLAG expression.

[00148] Expression and Purification of Recombinant Rat NELL2 Protein. For production of the C-terminally FLAG-tagged NELL2 peptide by insect cells. A
pIZT-NELL1-FLC plasmid was constructed by inserting the rat NELL2 cDNA fused to a FLAG epitope sequence derived from the pTB701-NELL2-FLC plasmid into insect expression vector pIZT/V5-His (Invitrogen). High Five cells were purchased from Invitrogen, and were cultured in High Five Serum-Free Medium (Invitrogen).
High Five cells were transfected with the pIZT-NELL1-FLC plasmid using FuGene6 (Roche). Forty-eight hours after transfection, cells were selected with 400 mg/ml of Zeocin (Invitrogen). Selective media was replaced every 3 to 4 days, until the stable expression cell line was established. NELL2 expression was confirmed in culture medium was confirmed using immunoprecipitation and Western blot analyses. High five cells were found to express NELL2 peptides (140-kDa) in the culture medium.

[00149] The recombinant rat NELL2-FLC peptide was purified from the culture medium of Zeocin-resistant High Five cells by anion exchange chromatography using a UNO Q-1 column (Bio-Rad). NELL2-FLC peptide was eluted at 500 mM
NaCI.

[00150] Example 2. Purification of NELL2 Protein From Culture Medium.

[00151] High Five cells carrying pIZT-FLC-NELL2 were cultured for about three days in serum free culture medium (1 L). The culture medium was centrifuged at.
3000 x g for 5 minutes and the supernatant was collected. PMSF was added to a final concentration of 1 mM. Saturated ammonium sulfate solution (80%
saturation (v/v) was added and the solution kept at 4 degrees for 1 hour. The solution was centrifuged at 15000 x g for 30 min. and precipitate collected. Precipitate was dissolved in 50 ml of 20 mM Tris-HC1 (pH 8.0), 1 mm EDTA at 4 degree and applied onto an anion-exchange chromatography UnoQ column (6 ml, Bio-Rad) equilibrated in 20 mM Tris-HC1 (pH 8.0), 1 mM EDTA at 4 degree (1 ml/min speed by FPLC (Amersham-Pharmacia). The column was thoroughly washed with the same buffer.
2s [00152] The binding protein was then eluted by the gradation from 0 M to 1.5 M
NaCI in the same buffer. The NELL2-FLAG fractions were identified by Western blotting using anti-Flag M2 (Sigma) Ab. The positive fractions were collected into one tube. Final product was dialyzed in the seamless cellulose tube (Vl/ako, cutoff MW 12000) against 1 L PBS for overnight at 4 degree. The product was stored at -70 degree.

[00153) The purity of the NELL2-FLAG peptide was examined by SDS-PAGEICBB staining. Fig. 18 is an illustration of a CBB-stained SDS-PAGE gel of UnoQ-eluate containing purified NELL2 peptide. Column A depicts a peptide band at about 130 kDa was isolated from the cell medium. "IP" refers to the Anti-FLAG
antibody used for the immunoprecipitation; "WB" refers to the Anti-FLAG
antibody used for the Western blotting detection.

[00154] Fig. 19 is a blot illustrating the expression of NELL1 and NELL2 from Five SFM. "ESF921" refers to a commercial name of a serum-free medium; "Five SFM"
refers to a commercial name of a medium. The constructs for the expression of both NELL proteins are similar to those described above.

[00155] Example 3. Increases in alkaline phosphatase activity is an early cellular marker of osteoblastic differentiation. In one study, fetal rat calvarial cells were grown in the presence of: NELL1 (1 ng/ml, 10ng/ml, 100ng/ml) produced using the methods described herein, or BMP4 (100ng/ml) for duration of time. Alkaline phosphatase was assayed in each sample by conventional methods.

[00156] Fig. 20 is a bar graph depicting alkaline phosphatase induction as a function of treatment in rat calvarial cell cultures ("OD" = Optic density).
Therefore, treatment with NELL1 was more potent than BMP4 in inducing osteoblast differentiation, as measured by alkaline phosphatase induction.

[00157) Fig. 21 are photomicrographs of rat calvarial cell cultures treated with NELL1. Treatment with NELL1 induced ,alkaline phosphatase activity and cell micronodule formation in the absence of ascorbic acid, which is an indication of osteoblastic differentiation and a precursor to bone formation.

[00158] Example 4. Alkaline phosphatase assay is an early cellular marker of osteoblastic difFerentiation. In one study, rat calvarial osteoblasts were grown on a 24 well plate. Wells were divided into groups including: NELL1, BMP2, NELL1/BMP2 and control (no peptide). Treatments included the application peptides at 100ng/ml. Alkaline phosphatase was assayed in.each sample by conventional methods.

[00159] Table 1.
Time NELL1 v~ BM_P2 .~''..NELL1IBMP Control 24 hr 134% 159% 210% 100%

3 days 154% 145% 189% 100%

[00'I60] Therefore, NELL1 and BMP have an additive effect on osteoblast differentiation, as measured by alkaline phosphatase activity relative to control or cells treated with single peptides alone.
[00161] Example 5. To investigate the effect of NELL1 expression on osteoblastic differentiation, bone related gene expression was evaluated in a microarray of MC3T3 cells at 3, 6 and 9 days post-infection with a NELL1 expressing construct relative to cells infected with a-gal expressing constructs.
[00162] Table 2. Expression levels over control cells.
Day 3 , , Day g ,. % Day 9 , . ' post infectionpost-infection ost infection , : , ~

Up regulated NA Osteocalcin 2.5 Decorin 2.2 . BMP7 2.1 Osteocalcin 2.6 Laminin B1 2.0 BMP7 3.2 Osteopontin 3.5 Col 15alpha1 2.6 ~uums~ Several bone related genes in NELL1 transfected cells were expressed at levels at least two fold higher than the a-gal control transfected cells.
Therefore, since cellular markers of late osteoblastic differentiation (such as osteocalcin and osteoponin) are up regulated, NELL1 expression and production enhanced osteoblastic differentiation.
[00164] Example 6. Micronodule formation, or the aggregation of a plurality of osteoblasts is an indication of osteoblastic differentiation and a precursor to bone formation. The process is thought to be regulated by ascorbic acid.
[00165] To investigate the effects of NELL1 on micronodule formation, MC3TC
cells were transfected with a NELL1 encoding construct, and grown in the absence of ascorbic acid.

[00166] Figs. 22 A&B are photomicrograph of MC3TC cells expressing NELL1 forming micronodules and stained for alkaline phosphatase (B). NELL1 expression induced alkaline phosphate induction, as well and micronodule formation.
Therefore, NELL1 is active in cell micronodule formation, which is a precursor to bone formation, and NELL1 alone is sufficient to induce osteoblast differentiation.
[00167] Example 7. Mineralization, or the intracellular accumulation of calcium is an indication of osteoblastic differentiation and a precursor to bone formation. To investigate the effects of NELL1 mineralization, primary calvarial cells were transfected with an adenoviral NELL1 encoding construct or a control construct encoding a-gal, or an antisense NELL1 virus. Cells were subsequently examined by Von Kassa staining to detect the presence of intracellular calcium accumulation after 3, 6, 9 and 12 days in culture. This demonstrates NELL1 can accelerate bone mineralization.
[00168] Figs. 23A-C are photomicrographs of calvarial cells treated with the A) antisense NELL1 virus, B) ~3-gal or C) NELL1. The control cells had .a moderate amount of mineralization, NELL1 expressing cells had increased levels of mineralization, and in antisense NELL1 cells mineralization was inhibited.
This "knock-out" study shows that NELL1 is required for osteoblast differentiation.
[00169] Figs. 23 D&E are bar graphs depicting osteocalcin and osteoponin mRNA expression as a ratio relative to control GAPFH, after 3, 6, 9 and 12 days in culture. NELL1 expressing cells expressed significantly elevated levels of osteocalcin and osteoponin mRNA after 12 days. Therefore, NELL1 is active in inducing the expression of late cellular markers of osteoblastic differentiation and mineralization, which is a precursor to bone formation.
[00170] Example 8. Transgenic animal models have been used to examine the effect of NELL1 over expression on bone formation. CMV promoter was linked to NELL1 cDNA and microinjected into fertilized eggs. NELL1 was pan-over-expressed under potent CMV promoter.
[00171] Fig. 24 is a photomicrograph of a NELL1 transgenic mouse tissue, depicting Von Kassa staining. As shown, in Fig. 24 NELL1 transgenic mice had calvarial overgrowth, confirming NELL1's ability to induce bone growth including membranous bone formation.
2s [00172] Fig. 25 A&B are photomicrographs depicting Von Kassa staining of calvaria of a NELL1 transgenic mouse (A) and normal littermate (B). As shown in Fig. 25A, NELL1 transgenic mice had enhanced mineralization relative to the normal littermate confirming NELL1's role in membranous bone formation.
[00173] Example 9. Transgenic animal models have been used to examine the effect of NELL1 expression on Cbfa1 deficiency induced developmental defects.
[00174] To determine whether Cbfa1 may play a role in NELL1 regulation, fetal rat calvarial cells were transfected with plasmid vectors containing mouse Cbfa1.
[00175] Fig. 26 is a blot depicting expression of NELL1 in Cbfa1 transfected cells at 24 and 48 hours relative to control cells. Cbfa1 transfection up regulated expression within 24 hours (along either positive control osteocalcin). This shows NELL-1 is downstream of Cbfa1 - a key "osteoblast transcription factor".
[00176] Fig. 27A-C are photographs of skeletal staining (top, middle) and micro-CT (bottom). Fig. 27A depicts the normal skeletal pattern of a wild-type mouse.
Typical boarders of mineralization are noted (dashed lines), anterior and posterior fontenelles (asterisks), and outline of the right coronal suture can be seen (arrows).
Also, a normal clavicle is shown (A-middle). The micro-CT reveals the typical craniofacial bone morphology. Fig. 27B depicts skeletal defects of a Cbfa1+~-animal. Specifically, defective bone mineralization and bone formation is present in the poorly stained tissue (between the dotted lines) lateral to the midline calvarial defect, and lucency can also be seen in the area of the coronal structure (arrows).
A significant degree of clavicle hypoplasia is noted (B-middle). Fig. DC
depicts skeletal defects of a Cbfa1+~-+ NELL1°"ereXP animal demonstrating significantly increased calvarial bone formation relative to the Cbfa1+~- haploid deficient animal on skeletal staining and micro-CT. Also, a significantly lesser degree of clavicle hypoplasia relative to the Cbfa1+~- haploid deficient animal (middle). Note the restoration of bony overlap at the coronal sutures (arrows). Therefore, NELL1 over expression rescued Cbfa1 deficiency in transgenic mice confirming NELL1's role in membranous bone formation and endochondral bone formation. Further, NELL 1 can regenerate bone in bone in birth defects.
[00177] Example 10. Critical size defect is an important model for the study of an agents ability to induce intramembraneous bone repair. To investigate the effects of NELL1 on bone repair, right and left calvarial defects (3mm) were created in wild-type adult CD-1 male mice. Left defects (control) were grafted with a PLGA/collagen carrier membrane only while right defects were grafted with PLGA/collagen carrier membrane soaked in either 200ng of NELL1 or BMP2 per site. Calvaria were extracted and examined by microCT analysis.
[00178] Fig. 28A is a photograph of control (left) and BMP2 (right) treatment of calvarial defect; is a photograph of control (left) and NELL1 (right) treatment of calvarial defect; Fig. 29 is a photograph of NELL1 (left) and BMP2 (right) treatment of calvarial defect. Significant amount of bone formation was observed in both NELL1 and BMP2 groups. Therefore, NELL1 expression significantly effected bone formation and induce bone regeneration in the critical size defect model confirming NELL1's role in membranous bone formation.
[00179] Example 11. Rapid Palatal Expansion (RPE) is another model for the study of an agents ability to induce intramembraneous bone repair. To investigate the effects of NELL1 on bone repair, 4-week old Sprague Dawley rats were divided into groups for 1 ) control expansion, and 2) expansion with NELL1 treatment.
The rats were sacrificed and their palates extracted an kept vital in organ culture. The palates were expanded and NELL1 added to the treatment group for 9 days.
[00180] Fig. 30A&B are photographs of expanded palates treated with NELL1 (A) and control (B). Significant amount of bone formation was observed in both and BMP2 groups. Therefore, NELL1 treatment significantly effected bone formation in the RPE model confirming NELL1's role in membranous bone formation.
[00181] Example 12. Endochondral bone formation is the key process in long bone development. It has several stages including: chondroblast proliferation, hypertrophy, apoptosis, invasion of blood vessel, replacement by osteoblasts.
Acceleration of any one of these stages will induce endochrondral bone growth.
[00182] Fig. 31A&B are photomicrographs of cartilage with TUNEL staining for apoptotic cells in NELL1 over expressing transgenic mice (A) and wild type mice (B).
[00183] As shown in Fig. 31A, in NELL1 over-expression in mice, cartilage shows hypertrophic chondroblasts and apoptosis (indicated by the brown staining using TUNEL ASSAY for identifying shrinkage of apoptotic nuclei). In Fig. 31 B is a normal mouse (wild type) cartilage with TUNEL staining very few apoptotic cells are present and the cells are not hypertrophic. Therefore, NELL1 can induce cartilage hypertrophy and apoptosis, thereby inducing long bone formation and regeneration.
[00184] Example 13. NELL substrate preparation.
[00185] In vitro. Polylactide-co-glycolide (85:15 PLGA; intrinsic viscosity ~0.6 dL/g, Birmingham Polymers, AL) was dissolved in chloroform to prepare 5%
solution and poured into glass culture dishes and allowed to slowly evaporate for 24 hours. After solvent extraction, the films were coated according to the 8 groups below: (a) polymer only with no coating; (b) conventional apatite (1 x SBF
followed by 1.5x SBF); (c) accelerated biomimetic apatite (5 x SBF followed by Mg-free and carbonate-free 5x SBF); (d) fibronectin (0.01 mg/ml); (e) poly-L-lysine (0.01 mg/ml);
(f) collagen; (g) Mefp-1 (0.01 mg/ml); and (h) mixture of collagen &
hyaluronan.
Each group was subdivided into NELL1 containing (100 ng) and NELL1-free groups, and cultured in vitro for 7 days with primary osteoblasts in non-differentiation media (no ascorbic acid, no beta glycerol phosphates). For each material, NELL1 groups stimulated higher alkaline phosphatase. activity than NELL1 counterparts.
Among the materials, accelerated apatites (group c) induced the greatest, and polymer control (group a) induced the least alkaline phosphatase activities.
[00186] In vivo. Polylactide-co-glycolide (85:15 PLGA; intrinsic viscosity ~0.6 dL/g, Birmingham Polymers, AL) was dissolved in chloroform and mixed with porogens (sucrose granules with diameter ~100-300,~m) to produce ~ 90%
porosity PLGA scaffolds after particulate leaching and solvent extraction.
Porous scaffolds were argon-plasma-etched, sterilized, coated with aqueous bovine type I
collagen mixture containing 200 ng NELL1 peptide, dried, and implanted into calvarial defects of adult male wild-type mice. Positive control (PLGA/collagen/BMP) and negative controls (PLGA/collagen only; no growth factors), were also implanted into similar defects. At 4 week, microCT
analysis show that while little or no bone formation was induced by the negative control scaffolds (PLGA/collagen only), NELL1-containing and BMP-containing scaffolds induced rapid and complete mineralization across the defects by week 4.
Conventional histology confirmed that the mineralization presents the classic features of mature bone.

[00187] It.is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. It will be understood that the invention may also comprise any combination of the embodiments described or combination with known methods and compositions.
[00188] Although now having described certain embodiments of NELL peptide expression systems and bone formation activity of NELL peptide, it is to be understood that the concepts implicit in these embodiments may be used in other embodiments as well. In short, the protection of this application is limited solely to the claims that now follow.

SEQUENCE LISTING
<110> The Regents of the University of California Ting, Kang Kuroda, Shunichi Wu, Ben <120> Nell Peptide Expression Systems and Bone Formation Activity of Nell Peptide <130> 38586-332 <150> US 60/445,672 <151> 2003-02-07 <160> 14 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 2433 <212> DNA
<213> Homo Sapiens <220>
<221> CDS
<222> (1)...(2433) <400>
1 atggatttg atttta gttgtgtgg ttctgtgtg tgcactgcc 48 atg ccg Met ProMetAspLeu IleLeu ValValTrp PheCysVal CysThrAla agg acagtggtgggc tttggg atggaccct gaccttcag atggatatc 96 Arg ThrValValGly PheGly MetAspPro AspLeuGln MetAspIle gtc accgagcttgac cttgtg aacaccacc cttggagtt getcaggtg 144 Val ThrGluLeuAsp LeuVal AsnThrThr LeuGlyVal AlaGlnVal tct ggaatgcacaat gccagc aaagcattt ttatttcaa gacatagaa 192 Ser GlyMetHisAsn AlaSer LysAlaPhe LeuPheGln AspIleGlu aga gagatccatgca getcct catgtgagt gagaaatta attcagctg 240 Arg GluIleHisAla AlaPro HisValSer GluLysLeu IleGlnLeu ttc cagaacaagagt gaattc accattttg gccactgta cagcagaag 288 Phe GlnAsnLysSer GluPhe ThrIleLeu AlaThrVal GlnGlnLys _ 2 _ cca tcc act tca gga gtg ata ctg tcc att cga gaa ctg gag 336 cac agc Pro Ser Thr Ser Gly Val Ile Leu Ser Ile Arg Glu Leu Glu His Ser tat ttt gaa ctg gag agc agt ggc ctg agg gat gag att cgg 384 tat cac Tyr Phe Glu Leu Glu Ser Ser Gly Leu Arg Asp Glu Ile Arg Tyr His tac ata cac aat ggg aag cca agg aca gag gca ctt cct tac 432 cgc atg Tyr Ile His Asn Gly Lys Pro Arg Thr Glu Ala Leu Pro Tyr Arg Met gca gat gga caa tgg cac aag gtt gca ctg tca gtt agc gcc 480 tct cat Ala Asp Gly Gln Trp His Lys Val Ala Leu Ser Val Ser Ala Ser His ctc ctg ctc cat gtc gac tgt aac agg att tat gag cgt gtg 528 ata gac Leu Leu Leu His Val Asp Cys Asn Arg Ile Tyr Glu Arg Val Ile Asp cct cca gat acc aac ctt CCC CCa gga atc aat tta tgg ctt 576 ggc cag Pro Pro Asp Thr Asn Leu Pro Pro Gly Ile Asn Leu Trp Leu Gly Gln cgc aac caa aag cat ggc tta ttc aaa ggg atc atc caa gat 624 ggg aag Arg Asn Gln Lys His Gly Leu Phe Lys Gly Ile Ile Gln Asp Gly Lys atc atc ttt atg ccg aat gga tat ata aca cag tgt cca aat 672 cta aat Ile Ile Phe Met Pro Asn Gly Tyr Ile Thr Gln Cys Pro Asn Leu Asn cac act tgc cca acc tgc agt gat ttc tta agc ctg gtg caa 720 gga ata His Thr Cys Pro~Thr Cys Ser Asp Phe Leu Ser Leu Val Gln Gly Ile atg gat tta caa gag ctt ttg gcc aag atg act gca aaa cta 768 aat tat Met Asp Leu Gln Glu Leu Leu Ala Lys Met Thr Ala Lys Leu Asn Tyr gca gag aca aga ctt agt caa ttg gaa aac tgt cat tgt gag 816 aag act Ala Glu Thr Arg Leu Ser Gln Leu Glu Asn Cys His Cys Glu Lys Thr tgt caa gtg agt gga ctg ctc tat cga gat caa gac tct tgg 864 gta gat Cys Gln Val Ser Gly Leu Leu Tyr Arg Asp Gln Asp Ser Trp Val Asp ggt gac cat tgc agg aac tgc act tgc aaa agt ggt gcc gtg 912 gaa tgc Gly Asp His Cys Arg Asn Cys Thr Cys Lys Ser Gly Ala Val Glu Cys a agg atg tcc tgt CCC CCt ctc aat tgc tcc cca gac tcc 960 ctc cca c g Arg Arg Met Ser Cys Pro Pro Leu Asn Cys Ser Pro Asp Ser Leu Pro gta cac att get ggc cag tgc tgt aag gtc tgc cga cca aaa 1008 tgt atc Val His Ile Ala Gly Gln Cys Cys Lys Val Cys Arg Pro Lys Cys Ile tat gga gga aaa gtt ctt gca gaa ggc cag cgg att tta acc 1056 aag agc Tyr Gly Gly Lys Val Leu Ala Glu Gly Gln Arg Ile Leu Thr Lys Ser tgt cgg gaa tgc cga ggt gga gtt tta gta aaa att aca gaa 1104 atg tgt Cys Arg Glu Cys Arg Gly Gly Val Leu Val Lys Ile Thr Glu Met Cys cct cct ttg aac tgc tca gaa aag gat cac att ctt cct gag 1152 aat cag Pro Pro Leu Asn Cys Ser Glu Lys Asp His Ile Leu Pro Glu Asn Gln tgc tgc cgt gtc tgt aga ggt cat aac ttt tgt gca gaa gga 1200 cct aaa Cys Cys Arg Val Cys Arg Gly His Asn Phe Cys Ala Glu Gly Pro Lys tgt ggt gaa aac tca gag tgc aaa aac tgg aat aca aaa get 1248 act tgt Cys Gly Glu Asn Ser Glu Cys Lys Asn Trp Asn Thr Lys Ala Thr Cys tgc aag agt ggt tac atc tct gtc cag gga gac tct gcc tac 1296 tgt a g g Glu Cys Lys Ser Gly Tyr Ile Ser Val Gln Gly Asp Ser Ala Tyr Cys aa gat att gat gag tgt gca get aag atg cat tac tgt cat 1344 gcc aat g Glu Asp Ile Asp Glu Cys Ala Ala Lys Met His Tyr Cys His Ala Asn act gtg tgt gtc aac ctt cct ggg tta tat cgc tgt gac tgt 1392 gtc cca Thr Val Cys Val Asn Leu Pro Gly Leu Tyr Arg Cys Asp Cys Val Pro a tac att cgt gtg gat gac ttc tct tgt aca gaa cac gat 1440 gaa tgt gg Gly Tyr Ile Arg Val Asp Asp Phe Ser Cys Thr Glu His Asp Glu Cys ggc agc ggc cag cac aac tgt gat gag aat gcc atc tgc acc 1488 aac act Gly Ser Gly Gln His Asn Cys Asp Glu Asn Ala Ile Cys Thr Asn Thr gtc cag gga cac agc tgc acc tgc aaa ccg ggc tac gtg ggg 1536 aac ggg Val Gln Gly His Ser Cys Thr Cys Lys Pro Gly Tyr Val Gly Asn Gly accatctgc agaget ttctgtgaagag ggctgcaga tacggtgga acg 1584 ThrIleCys ArgAla PheCysGluGlu GlyCysArg TyrGlyGly Thr tgtgtgget cccaac aaatgtgtctgt ccatctgga ttcacagga agc 1632 CysValAla ProAsn LysCysValCys ProSerGly PheThrGly Ser cactgcgag aaagat attgatgaatgt tcagaggga atcattgag tgc 1680 HisCysGlu LysAsp IleAspGluCys SerGluGly IleIleGlu Cys cacaaccat tcccgc tgcgttaacctg ccagggtgg taccactgt gag 1728 HisAsnHis SerArg CysValAsnLeu ProGlyTrp TyrHisCys Glu tgcagaagc ggtttc catgacgatggg acctattca ctgtccggg gag 1776 CysArgSer GlyPhe HisAspAspGly ThrTyrSer LeuSerGly Glu tcctgtatt gacatt gatgaatgtgcc ttaagaact cacacctgt tgg 1824 SerCysIle AspIle AspGluCysAla LeuArgThr HisThrCys Trp aacgattct gcctgc atcaacctggca gggggtttt gactgtctc tgc 1872 AsnAspSer AlaCys IleAsnLeuAla GlyGlyPhe AspCysLeu Cys ccctctggg ccctcc tgctctggtgac tgtcctcat gaagggggg ctg 1920 ProSerGly ProSer CysSerGlyAsp CysProHis GluGlyGly Leu aagcacaat ggccag gtgtggaccttg aaagaagac aggtgttct gtc 1968 LysHisAsn GlyGln ValTrpThrLeu LysGluAsp ArgCysSer Val tgctcctgc aaggat ggcaagatattc tgccgacgg acagettgt gat 2016 CysSerCys LysAsp GlyLysIlePhe CysArgArg ThrAlaCys Asp tgc cag aat cca agt get gac cta ttc tgt tgc eca gaa tgt gac acc 2064 Cys Gln Asn Pro Ser Ala Asp Leu Phe Cys Cys Pro Glu Cys Asp Thr aga gtc aca agt caa tgt tta gac caa aat ggt cac aag ctg tat cga 2112 Arg Val Thr Ser Gln Cys Leu Asp Gln Asn Gly His Lys Leu Tyr Arg agt gga gac aat tgg acc cat agc tgt cag cag tgt cgg tgt ctg gaa 2160 Ser Gly Asp Asn Trp Thr His Ser Cys Gln Gln Cys Arg Cys Leu Glu gga gag gta gat tgc tgg cca ctc act tgc ccc aac ttg agc tgt gag 2208 Gly Glu Val Asp Cys Trp Pro Leu Thr Cys Pro Asn Leu Ser Cys Glu tatacagetatc ttagaaggg gaatgttgt CCCCgCtgt gtcagt gac 2256 TyrThrAlaIle LeuGluGly GluCysCys ProArgCys ValSer Asp ccctgcctaget gataacato acctatgao atcagaaaa acttgc ctg 2304 ProCysLeuAla AspAsnIle ThrTyrAsp IleArgLys ThrCys Leu gacagctatggt gtttcacgg cttagtggc tcagtgtgg acgatg get 2352 AspSerTyrGly ValSerArg LeuSerGly SerValTrp ThrMet Ala ggatctccctgc aoaaoctgt aaatgcaag aatggaaga gtotgt tgt 2400 GlySerProCys ThrThrCys LysCysLys AsnGlyArg ValCys Cys totgtggatttt gagtgtctt caaaataat tga 2433 SerValAspPhe GluCysLeu GlnAsnAsn <210> 2 <211> 810 <212> PRT
<213> Homo sapiens <400> 2 Met Pro Met Asp Leu Ile Leu Val Val Trp Phe Cys Val Cys Thr Ala Arg Thr Val Val Gly Phe Gly Met Asp Pro Asp Leu Gln Met Asp Ile Val Thr Glu Leu Asp Leu Val Asn Thr Thr Leu Gly Val Ala Gln Val Ser Gly Met His Asn Ala Ser Lys Ala Phe Leu Phe Gln Asp Ile Glu Arg Glu Ile His Ala Ala Pro His Val Ser Glu Lys Leu Ile Gln Leu Phe Gln Asn Lys Ser Glu Phe Thr I1e Leu Ala Thr Val Gln Gln Lys Pro Ser Thr Ser Gly Val Ile Leu Ser Ile Arg Glu Leu Glu His Ser Tyr Phe Glu Leu Glu Ser Ser Gly Leu Arg Asp Glu Ile Arg Tyr His Tyr Ile His Asn Gly Lys Pro Arg Thr Glu Ala Leu Pro Tyr Arg Met Ala Asp Gly Gln Trp His Lys Val Ala Leu Ser Val Ser Ala Ser His Leu Leu Leu His Val Asp Cys Asn Arg Ile Tyr Glu Arg Val Ile Asp Pro Pro Asp Thr Asn Leu Pro Pro Gly Ile Asn Leu Trp Leu Gly Gln Arg Asn Gln Lys His Gly Leu Phe Lys Gly Ile Ile Gln Asp Gly Lys Ile Ile Phe Met Pro Asn Gly Tyr Ile Thr Gln Cys Pro Asn Leu Asia.

His Thr Cys Pro Thr Cys Ser Asp Phe Leu Ser Leu Val Gln Gly Ile Met Asp Leu Gln Glu Leu Leu Ala Lys Met Thr Ala Lys Leu Asn Tyr Ala Glu Thr Arg Leu Ser Gln Leu Glu Asn Cys His Cys Glu Lys Thr Cys Gln Val Ser Gly Leu Leu Tyr Arg Asp Gln Asp Ser Trp Val Asp Gly Asp His Cys Arg Asn Cys Thr Cys Lys Ser Gly Ala Val Glu Cys Arg Arg Met Ser Cys Pro Pro Leu Asn Cys Ser Pro Asp Ser Leu Pro Val His Ile Ala Gly Gln Cys Cys Lys Val Cys Arg Pro Lys Cys Ile Tyr Gly Gly Lys Val Leu Ala Glu Gly Gln Arg Ile Leu Thr Lys Ser Cys Arg Glu Cys Arg Gly Gly Val Leu Val Lys Ile Thr Glu Met Cys Pro Pro Leu Asn Cys Ser Glu Lys Asp His Ile Leu Pro Glu Asn Gln Cys Cys Arg Val Cys Arg Gly His Asn Phe Cys Ala Glu Gly Pro Lys Cys Gly Glu Asn Ser Glu Cys Lys Asn Trp Asn Thr Lys Ala Thr Cys Glu Cys Lys Ser Gly Tyr Ile Ser Val Gln Gly Asp Ser Ala Tyr Cys Glu Asp Ile Asp Glu Cys Ala Ala Lys Met His Tyr Cys His Ala Asn Thr Val Cys Val Asn Leu Pro Gly Leu Tyr Arg Cys Asp Cys Val Pro Gly Tyr Ile Arg Val Asp Asp Phe Ser Cys Thr Glu His Asp Glu Cys Gly Ser Gly Gln His Asn Cys Asp Glu Asn Ala Ile Cys Thr Asn Thr Val Gln Gly His Ser Cys Thr Cys Lys Pro Gly Tyr Val Gly Asn Gly Thr Ile Cys Arg Ala Phe Cys Glu Glu Gly Cys Arg Tyr Gly Gly Thr 515 520 525 , Cys Val Ala Pro Asn Lys Cys Val Cys Pro Ser Gly Phe Thr Gly Ser His Cys Glu Lys Asp Ile Asp Glu Cys Ser Glu Gly Ile Ile Glu Cys His Asn His Ser Arg Cys Val Asn Leu Pro Gly Trp Tyr His Cys Glu Cys Arg Ser Gly Phe His Asp Asp Gly Thr Tyr Ser Leu Ser Gly Glu Ser Cys Ile Asp Ile Asp Glu Cys Ala Leu Arg Thr His Thr Cys Trp _ 7 _ Asn Asp Ser Ala Cys Ile Asn Leu Ala Gly Gly Phe Asp Cys Leu Cys Pro Ser Gly Pro Ser Cys Ser Gly Asp Cys Pro His Glu Gly Gly Leu Lys His Asn Gly Gln Val Trp Thr Leu Lys Glu Asp Arg Cys Ser Val Cys Ser Cys Lys Asp Gly Lys Ile Phe Cys Arg Arg Thr Ala Cys Asp Cys Gln Asn Pro Ser Ala Asp Leu Phe Cys Cys Pro Glu Cys Asp Thr Arg Val Thr Ser Gln Cys Leu Asp Gln Asn Gly His Lys Leu Tyr Arg Ser Gly Asp Asn Trp Thr His Ser Cys Gln Gln Cys Arg Cys Leu Glu Gly Glu Val Asp Cys Trp Pro Leu Thr Cys Pro Asn Leu Ser Cys Glu Tyr Thr Ala Ile Leu Glu Gly Glu Cys Cys Pro Arg Cys Val Ser Asp Pro Cys Leu Ala Asp Asn Ile Thr Tyr Asp Ile Arg Lys Thr Cys Leu Asp Ser Tyr Gly Val Ser Arg Leu Ser Gly Ser Val Trp Thr Met Ala Gly Ser Pro Cys Thr Thr Cys Lys Cys Lys Asn Gly Arg Val Cys Cys Ser Val Asp Phe Glu Cys Leu Gln Asn Asn <210> 3 <211> 2433 <212> DNA
<213> Rattus norvegicus <220>
<221> CDS
<222> (1)...(2433) <400> 3 atg ccg atg gat gtg att tta gtt ttg tgg ttc tgt gta tgc acc gcc 48 Met Pro Met Asp Val Ile Leu Val Leu Trp Phe Cys Val Cys Thr Ala agg aca gtg ttg ggc ttt ggg atg gac cct gac ctt cag ctg gac atc 96 Arg Thr Val Leu Gly Phe Gly Met Asp Pro Asp Leu Gln Leu Asp Ile atc tca gag ctc gac ctg gtg aac acc acc ctg gga gtc acg cag gtg 144 Ile Ser Glu Leu Asp Leu Val Asn Thr Thr Leu Gly Val Thr Gln Val get gga ctg cac aac gcc agt aaa gca ttt cta ttt caa gat gta cag 192 Ala Gly Leu His Asn Ala Ser Lys Ala Phe Leu Phe Gln Asp Val Gln _ g _ aga gag atc cat tcg gcc cct cac gtg agt gag aag ctg atc cag cta 240 Arg Glu Ile His Ser Ala Pro His Val Ser Glu Lys Leu Ile Gln Leu ttc cgg aat aag agc gag ttc acc ttt ttg get aca gtg cag cag aaa 288 Phe Arg Asn Lys Ser Glu Phe Thr Phe Leu Ala Thr Val Gln Gln Lys cca tcc acc tca ggg gtg ata ctg tcc atc egg gag ctg gag cac agc 336 Pro Ser Thr Ser Gly Val Ile Leu Ser Ile Arg Glu Leu G1u His Ser tat ttt gaa ctg gag agc agt ggc cca aga gaa gag ata cgc tac cat 384 Tyr Phe Glu Leu Glu Ser Ser Gly Pro Arg Glu Glu Ile Arg Tyr His tacatacatggt ggaaag cccaggactgag gccctt ccctaccgcatg 432 TyrIleHisGly GlyLys ProArgThrGlu AlaLeu ProTyrArgMet gcagacggacaa tggcac aaggtcgcgctg tcagtg agcgcctctcac 480 AlaAspGlyGln TrpHis LysValAlaLeu SerVal SerAlaSerHis l45 150 155 160 ctcctgctccac atcgac tgcaataggatt tacgag cgtgtgatagac 528 LeuLeuLeuHis IleAsp CysAsnArgIle TyrGlu ArgValIleAsp cctccggagacc aacctt cctccaggaagc aatctg tggcttgggcaa 576 ProProGluThr AsnLeu ProProGlySer AsnLeu TrpLeuGlyGln cgt aac caa aag cat ggc ttt ttc aaa gga atc atc caa gat ggt aag 624 Arg Asn Gln Lys His Gly Phe Phe Lys Gly Ile Ile Gln Asp Gly Lys atc atc ttc atg ccg aat ggt ttc atc aca cag tgt ccc aac ctc aat 672 Ile Ile Phe Met Pro Asn Gly Phe Ile Thr Gln Cys Pro Asn Leu Asn cgc act tgc cca aca tgc agt gac ttc ctg agc ctg gtt caa gga ata 720 Arg Thr Cys Pro Thr Cys Ser Asp Phe Leu Ser Leu Val Gln Gly Ile atg gat ttg caa gag ctt ttg gcc aag atg act gca aaa ctg aat tat 768 Met Asp Leu Gln Glu Leu Leu Ala'Lys Met Thr Ala Lys Leu Asn Tyr gca gag acg aga ctt ggt caa ctg gaa aat tgc cac tgt gag aag acc 816 Ala Glu Thr Arg Leu Gly Gln Leu Glu Asn Cys His Cys Glu Lys Thr tgc caa gtg agt ggg ctg ctc tac agg gac caa gac tcc tgg gtg gat 864 _ g _ Cys Gln Val Ser Gly Leu Leu Tyr Arg Asp Gln Asp Ser Trp Val Asp ggt gac aac tgt ggg aac tgc acg tgc aaa agt ggt gcc gtg gag tgc 912 Gly Asp Asn Cys Gly Asn Cys Thr Cys Lys Ser Gly Ala Val Glu Cys cgc agg atg tcc tgt ccc ccg ctc aac tgt tcc ccg gac tca ctt cct 960 Arg Arg Met Ser Cys Pro Pro Leu Asn Cys Ser Pro Asp Ser Leu Pro gtg cac att tcc ggc cag tgt tgt aaa gtt tgc aga cca aaa tgt atc 1008 Val His Ile Ser Gly Gln Cys Cys Lys Val Cys Arg Pro Lys Cys Ile tat gga gga aaa gtt ctt get gag ggc cag cgg att tta acc aag acc 1056 Tyr Gly Gly Lys Val Leu Ala Glu Gly Gln Arg Ile Leu Thr Lys Thr tgc cgg gaa tgt cga ggt gga gtc ttg gta aaa atc aca gaa get tgc 1104 Cys Arg Glu Cys Arg Gly Gly Val Leu Val Lys Ile Thr Glu Ala Cys cct cct ttg aac tgc tca gca aag gat cat att ctt cca gag aat cag 1152 Pro Pro Leu Asn Cys Ser Ala Lys Asp His Ile Leu Pro Glu Asn Gln tgc tgc agg gtc tgc cca ggt cat aac ttc tgt gca gaa gca cct aag 1200 Cys Cys Arg Val Cys Pro Gly His Asn Phe Cys Ala Glu Ala Pro Lys tgc gga gaa aac tcg gaa tgc aaa aat tgg aat aca aaa gca acc tgt 1248 Cys Gly Glu Asn Ser Glu Cys Lys Asn Trp Asn Thr Lys Ala Thr Cys gag tgc aag aat gga tac atc tct gtc cag ggc aac tct gca tac tgt 1296 Glu Cys Lys Asn Gly Tyr Ile Ser Val Gln Gly Asn Ser Ala Tyr Cys gaa gat att gat gag tgt gca get aaa atg cac tat tgt cat gcc aac 1344 Glu Asp Ile Asp Glu Cys Ala Ala Lys Met His Tyr Cys His Ala Asn acc gtg tgt gtc aac ttg ccg ggg ttg tat cgc tgt gac tgc gtc cca 1392 Thr Val Cys Val Asn Leu Pro Gly Leu Tyr Arg Cys Asp Cys Val Pro ggg tac atc cgt gtg gat gac ttc tct tgt acg gag cat gat gat tgt 1440 Gly Tyr Ile Arg Val Asp Asp Phe Ser Cys Thr Glu His Asp Asp Cys ggc agc gga caa cac aac tgc gac aaa aat gcc atc tgt acc aac aca 1488 Gly Ser Gly Gln His Asn Cys Asp Lys Asn Ala Ile Cys Thr Asn Thr gtc cag gga cac agc tgc acc tgc cag ccg ggt tac gtg gga aat ggc 1536 Val Gln Gly His Ser Cys Thr Cys Gln Pro Gly Tyr Val Gly Asn Gly acc atc tgc aaa gca ttc tgt gaa gag ggt tgc aga tac gga ggt acc 1584 Thr Ile Cys Lys Ala Phe Cys Glu Glu Gly Cys Arg Tyr Gly Gly Thr tgt gtg get cct aac aag tgt gtc tgt cct tct gga ttc acg gga agc 1632 Cys Val Ala Pro Asn Lys Cys Val Cys Pro Ser Gly Phe Thr Gly Ser cac tgt gag aaa gat att gat gaa tgc gca gag gga ttc gtt gaa tgc 1680 His Cys Glu Lys Asp Ile Asp Glu Cys Ala Glu Gly Phe Val Glu Cys cac aac tac tcc cgc tgt gtt aae ctg cca ggg tgg tac cac tgt gag 1728 His Asn Tyr Ser Arg Cys Val Asn Leu Pro Gly Trp Tyr His Cys Glu tgc aga agc ggt ttc cat gac gat ggg acc tac tca ctg tcc ggg gag 1776 Cys Arg Ser Gly Phe His Asp Asp Gly Thr Tyr Ser Leu Ser Gly Glu tcc tgc att gat atc gat gaa tgt gcc tta aga act cac act tgt tgg 1824 Ser Cys Ile Asp Ile Asp Glu Cys Ala Leu Arg Thr His Thr Cys Trp aat gac tct gcc tgc atc aac tta gca gga gga ttt gac tgc ctg tgt 1872 Asn Asp Ser Ala Cys Ile Asn Leu Ala Gly Gly Phe Asp Cys Leu Cys ccc tct ggg CCC tcc tgc tct ggt gac tgt CCC CaC gaa gga ggg ctg 1920 Pro Ser Gly Pro Ser Cys Ser Gly Asp Cys Pro His Glu Gly Gly Leu aag cat aat ggg cag gtg tgg att ctg aga gaa gac agg tgt tca gtc 1968 Lys His Asn Gly Gln Val Trp Ile Leu Arg Glu Asp Arg Cys Ser Val tgt tcc tgc aag gat ggg aag ata ttc tgc cgg cgg aca get tgt gat 2016 Cys Ser Cys Lys Asp Gly Lys Ile Phe Cys Arg Arg Thr Ala Cys Asp tgc cag aat cca aat gtt gac ctt ttt tgc tgc cca gag tgc gat acc 2064 Cys Gln Asn Pro Asn Val Asp Leu Phe Cys Cys Pro Glu Cys Asp Thr agg gtc acc agc caa tgt tta gat caa agt gga cag aag ctc tat cga 2112 Arg Val Thr Ser Gln Cys Leu Asp Gln Ser Gly Gln Lys Leu Tyr Arg agt gga gac aac tgg acc cac agc tgc cag cag tgc cga tgt ctg gaa 2160 Ser Gly Asp Asn Trp Thr His Ser Cys Gln Gln Cys Arg Cys Leu Glu gga gag gca gac tgc tgg cct ctg get tgc cct agt ttg ggc tgt gaa 2208 Gly Glu Ala Asp Cys Trp Pro Leu Ala Cys Pro Ser Leu Gly Cys Glu tac aca gcc atg ttt gaa ggg gag tgt tgt ccc cga tgt gtc agt gac 2256 Tyr Thr Ala Met Phe Glu Gly Glu Cys Cys Pro Arg Cys Val Ser Asp ccc tgc ctg get ggt aat att gcc tat gac atc aga aaa act tgc ctg 2304 Pro Cys Leu Ala Gly Asn Ile Ala Tyr Asp Ile Arg Lys Thr Cys Leu gac agc ttt ggt gtt tcg agg ctg agc gga gcc gtg tgg aca atg get 2352 Asp Ser Phe Gly Val Ser Arg Leu Ser Gly Ala Val Trp Thr Met Ala gga tct cct tgt aca acc tgc aaa tgc aag aat ggg aga gtc tgc tgc 2400 Gly Ser Pro Cys Thr Thr Cys Lys Cys Lys Asn Gly Arg Val Cys Cys tct gtg gat ctg gag tgt att gag aat aac tga 2433 Ser Val Asp Leu Glu Cys Ile Glu Asn Asn <210> 4 <211> 810 <212> PRT
<213> Rattus norvegicus <400> 4 Met Pro Met Asp Val Ile Leu Val Leu Trp Phe Cys Val Cys Thr Ala Arg Thr Val Leu Gly Phe Gly Met Asp Pro Asp Leu Gln Leu Asp Ile Ile Ser Glu Leu Asp Leu Val Asn Thr Thr Leu Gly Val Thr Gln Val Ala Gly Leu His Asn Ala Ser Lys Ala Phe Leu Phe Gln Asp Val Gln Arg Glu Ile His Ser Ala Pro His Val Ser Glu Lys Leu Ile Gln Leu Phe Arg Asn Lys Ser Glu Phe Thr Phe Leu Ala Thr Val Gln Gln Lys Pro Ser Thr Ser Gly Val Ile Leu Ser Ile Arg Glu Leu Glu His Ser Tyr Phe Glu Leu Glu Ser Ser Gly Pro Arg Glu Glu Ile Arg Tyr His Tyr Ile His Gly Gly Lys Pro Arg Thr Glu Ala Leu Pro Tyr Arg Met Ala Asp Gly Gln Trp His Lys Val Ala Leu Ser Val Ser Ala Ser His Leu Leu Leu His Ile Asp Cys Asn Arg Ile Tyr Glu Arg Val Ile Asp Pro Pro Glu Thr Asn Leu Pro Pro Gly Ser Asn Leu Trp Leu Gly Gln Arg Asn Gln Lys His Gly Phe Phe Lys Gly Ile Ile Gln Asp Gly Lys Ile Ile Phe Met Pro Asn Gly Phe Ile Thr Gln Cys Pro Asn Leu Asn Arg Thr Cys Pro Thr Cys Ser Asp Phe Leu Ser Leu Val Gln Gly Ile Met Asp Leu Gln Glu Leu Leu Ala Lys Met Thr Ala Lys Leu Asn Tyr Ala Glu Thr Arg Leu Gly Gln Leu Glu Asn Cys His Cys Glu Lys Thr Cys Gln Val Ser Gly Leu Leu Tyr Arg Asp Gln Asp Ser Trp Val Asp Gly Asp Asn Cys Gly Asn Cys Thr Cys Lys Ser Gly Ala Val Glu Cys Arg Arg Met Ser Cys Pro Pro Leu Asn Cys Ser Pro Asp Ser Leu Pro Val His Ile Ser Gly Gln Cys Cys Lys Val Cys~Arg Pro Lys Cys Ile Tyr Gly Gly Lys Val Leu Ala Glu Gly Gln Arg Ile Leu Thr Lys Thr Cys Arg Glu Cys Arg Gly Gly Val Leu Val Lys Ile Thr Glu Ala Cys Pro Pro Leu Asn Cys Ser Ala Lys Asp His Ile Leu Pro Glu Asn Gln Cys Cys Arg Val Cys Pro Gly His Asn Phe Cys Ala Glu Ala Pro Lys Cys Gly Glu Asn Ser Glu Cys Lys Asn Trp Asn Thr Lys Ala Thr Cys Glu Cys Lys Asn Gly Tyr Ile Ser Val Gln Gly Asn Ser Ala Tyr Cys Glu Asp Ile Asp Glu Cys Ala Ala Lys Met His Tyr Cys His Ala Asn Thr Val Cys Val Asn Leu Pro Gly Leu Tyr Arg Cys Asp Cys Val Pro Gly Tyr Ile Arg Val Asp Asp Phe Ser Cys Thr Glu His Asp Asp Cys Gly Ser Gly Gln His Asn Cys Asp Lys Asn Ala Ile Cys Thr Asn Thr Val Gln Gly His Ser Cys Thr Cys Gln Pro Gly Tyr Val Gly Asn Gly Thr Ile Cys Lys Ala Phe Cys Glu Glu Gly Cys Arg Tyr Gly Gly Thr Cys Val Ala Pro Asn Lys Cys Val Cys Pro Ser Gly Phe Thr Gly Ser His Cys Glu Lys Asp Ile Asp Glu Cys Ala Glu Gly Phe Val Glu Cys His Asn Tyr Ser Arg Cys Val Asn Leu Pro Gly Trp Tyr His Cys Glu Cys Arg Ser Gly Phe His Asp Asp Gly Thr Tyr Ser Leu Ser Gly Glu Ser Cys Ile Asp Ile Asp Glu Cys Ala Leu Arg Thr His Thr Cys Trp Asn Asp Ser Ala Cys Ile Asn Leu Ala Gly Gly Phe Asp Cys Leu Cys Pro Ser Gly Pro Ser Cys Ser Gly Asp Cys Pro His Glu Gly Gly Leu Lys His Asn Gly Gln Val Trp Ile Leu Arg Glu Asp Arg Cys Ser Val Cys Ser Cys Lys Asp Gly Lys Ile Phe Cys Arg Arg Thr Ala Cys Asp Cys Gln Asn Pro Asn Val Asp Leu Phe Cys Cys Pro Glu Cys Asp Thr Arg Val Thr Ser Gln Cys Leu Asp Gln Ser Gly Gln Lys Leu Tyr Arg Ser Gly Asp Asn Trp Thr His Ser Cys Gln Gln Cys Arg Cys Leu Glu Gly Glu Ala Asp Cys Trp Pro Leu Ala Cys Pro Ser Leu Gly Cys Glu Tyr Thr Ala Met Phe Glu Gly Glu Cys Cys Pro Arg Cys Val Ser Asp Pro Cys Leu Ala Gly Asn Ile Ala Tyr Asp Ile Arg Lys Thr Cys Leu Asp Ser Phe Gly Val Ser Arg Leu Ser Gly Ala Val Trp Thr Met Ala Gly Ser Pro Cys Thr Thr Cys Lys Cys Lys Asn Gly Arg Val Cys Cys Ser Val Asp Leu Glu Cys Ile Glu Asn Asn <210> 5 <211> 2433 <212> DNA
<213> Mus musculus <220>
<221> CDS
<222> (1)...(2433) <400> 5 atg ccg atg gat gtg att tta gtt ttg tgg ttc tgt gtg tgc acc gcc 48 Met Pro Met Asp Val Ile Leu Val Leu Trp Phe Cys Val Cys Thr Ala cag gac agt ggt ggg ctt tgg gat gga ccc tga cct tca gat gga cat 96 Gln Asp Ser Gly Gly Leu Trp Asp Gly Pro * Pro Ser Asp Gly His cat cac tga act tga cct tgt gaa cac cag ccc tgg gcg tca ctc agg 144 His His * Thr * Pro Cys Glu His Gln Pro Trp Ala Ser Leu Arg tgg gtg gac tac aca atg cca gta agg cat ttc tgt ttc aag atg tac 192 Trp Val Asp Tyr Thr Met Pro Val Arg His Phe Cys Phe Lys Met Tyr aga gag aga tcc act cag ccc ctc atg tga gtg aga agc tga tcc agc 240 Arg Glu Arg Ser Thr Gln Pro Leu Met * Val Arg Ser * Ser Ser tat tcc gga ata aga gtg agt tta cet ttt tgg cta cag tgc agc aga 288 Tyr Ser Gly Ile Arg Val Ser Leu Pro Phe Trp Leu Gln Cys Ser Arg agc cgt cca cct cag ggg tga tac tgt cga tcc ggg agc tgg aac aca 336 Ser Arg Pro Pro Gln Gly * Tyr Cys Arg Ser Gly Ser Trp Asn Thr getatt ttgaactggaga gcagtg gcccaagagaag agatac getatc 384 AlaIle LeuAsnTrpArg AlaVal AlaGlnGluLys ArgTyr AlaIle attaCa tccatggcggca agccca ggactgaggCCC ttCCCt aCCgCa 432 IleThr SerMetAlaAla SerPro GlyLeuArgPro PhePro ThrAla tggccg atggacagtggc acaagg tcgcgctgtctg tgagcg Cctctc 480 TrpPro MetAspSerGly ThrArg SerArgCysLeu * Ala ProLeu acctcc tactccatgtcg actgca ataggatttatg agcgtg tgatag 528 ThrSer TyrSerMetSer ThrAla IleGlyPheMet SerVal atcctc cggagaccaacc ttcctc caggaagcaatc tatggc ttgggc 576 IleLeu ArgArgProThr PheLeu GlnGluAlaIle TyrGly LeuGly aac gta atc aaa agc atg get ttt tca aag gaa tca tcc aag atg gca 624 Asn Val Ile Lys Ser Met Ala Phe Ser Lys Glu Ser Ser Lys Met Ala aga tca tct tca tgc cga acg get tca tca cac agt gcc cca acc taa 672 Arg Ser Ser Ser Cys Arg Thr Ala Ser Ser His Ser Ala Pro Thr atc gca ctt gcc caa cat gca gtg att tcc tga gcc tgg ttc aag gaa 720 Ile Ala Leu Ala Gln His Ala Val Ile Ser * Ala Trp Phe Lys Glu taa tgg att tgc aag agc ttt tgg cca aga tga ctg caa aac tga att 768 * Trp Ile Cys Lys Ser Phe Trp Pro Arg * Leu Gln Asn * Ile atg cag aga cga gac ttg gtc aac tgg aaa att gcc act gtg aga aga 816 Met Gln Arg Arg Asp Leu Val Asn Trp Lys Ile Ala Thr Val Arg Arg cct gcc aag tga gtg ggc tgc tct aca ggg acc aag act cct ggg tag 864 Pro Ala Lys * Val Gly Cys Ser Thr Gly Thr Lys Thr Pro Gly atggtgaca actgcagga actgcacat gcaaaagtg gtgctgtgg agt 912 MetValThr ThrAlaGly ThrAlaHis AlaLysVal ValLeuTrp Ser gccgaagga tgtcctgtc ccccactca actgttccc cagactcac ttc 960 AlaGluGly CysProVal ProHisSer ThrValPro GlnThrHis Phe ctgtgcata tttctggcc aatgttgta aagtttgca gaccaaaat gta 1008 LeuCysIle PheLeuAla AsnValVal LysPheAla AspGlnAsn Val tctatggag gaaaagttc ttgctgagg gccagcgga ttttaacca aga 1056 SerMetGlu GluLysPhe LeuLeuArg AlaSerGly Phe* Pro Arg CCtgCCggg aatgtcgag gtggagtct tggtaaaaa tcacagaag ctt 1104 ProAlaGly AsnValGlu ValGluSer Trp* Lys SerGlnLys Leu 340 3,45 350 gcc ctc ctt tga act get cag aga agg atc ata ttc ttc cgg aga acc 1152 Ala Leu Leu .* Thr Ala Gln Arg Arg Ile Ile Phe Phe Arg Arg Thr agt get ggg gtc tgc cga ggt cat aac ttc tgt gca gaa gca cct aag 1200 Ser Ala Gly Val Cys Arg Gly His Asn Phe Cys Ala Glu Ala Pro Lys tgt gga gaa aac tcg gaa tgc aaa aat tgg aat aca aaa gcg act tgt 1248 Cys Gly Glu Asn Ser Glu Cys Lys Asn Trp Asn Thr Lys Ala Thr Cys gag tgc aag aat gga tac atc tct gtc cag ggc aac tct gca tac tgt 1296 Glu Cys Lys Asn Gly Tyr Ile Ser Val Gln Gly Asn Ser Ala Tyr Cys gaa gat atc gat gag tgt gca gca aag atg cac tac tgt cat gcc aac 1344 Glu Asp Ile Asp Glu Cys Ala Ala Lys Met His Tyr Cys His Ala Asn acg gtg tgt gtc aac ttg ccg ggg tta tat cgc tgt gac tgc atc cca 1392 Thr Val Cys Val Asn Leu Pro Gly Leu Tyr Arg Cys Asp Cys Ile Pro gga tac atc cgt gtg gat gac ttc tct tgt acg gag cat gat gat tgt 1440 Gly Tyr Ile Arg Val Asp Asp Phe Ser Cys Thr Glu His Asp Asp Cys ggc agc gga caa cac aac tgt gac aaa aat gcc atc tgt acc aac aca 1488 Gly Ser Gly Gln His Asn Cys Asp Lys Asn Ala Ile Cys Thr Asn Thr gtc cag gga cac agc tgt acc tgc cag cca ggc tac gtg gga aat ggt 1536 Val Gln Gly His Ser Cys Thr Cys Gln Pro Gly Tyr Val Gly Asn Gly act gtc tgc aaa gca ttc tgt gaa gag ggt tgc aga tac gga ggt acc 1584 Thr Val Cys Lys Ala Phe Cys Glu Glu Gly Cys Arg Tyr Gly Gly Thr tgt gtg gcc cct aac aaa tgt gtc tgt cct tct gga ttc aca gga agc 1632 Cys Val Ala Pro Asn Lys Cys Val Cys Pro Ser Gly Phe Thr Gly Ser cac tgt gag aaa gat att gat gaa tgt gca gag gga ttc gtt gag tgc 1680 His Cys Glu Lys Asp Ile Asp Glu Cys Ala Glu Gly Phe Val Glu Cys cacaaccac tcccgctgc gttaacctt ccagggtgg taccactgt gag 1728 HisAsnHis SerArgCys ValAsnLeu ProGlyTrp TyrHisCys Glu tgcagaagc ggtttccat gacgatggg acctattca ctgtccggg gag 1776 CysArgSer GlyPheHis AspAspGly ThrTyrSer LeuSerGly Glu tcctgcatt gatattgat gaatgtgcc ttaagaact cacacttgt tgg 1824 SerCysIle AspIleAsp GluCysAla LeuArgThr HisThrCys Trp aatgactCt gCCtgcatc aacttagca ggaggattt gactgcctg tgt 1872 AsnAspSer AlaCysIle AsnLeuAla GlyGlyPhe AspCysLeu Cys ccctctggg ccctcctgc tctggtgac tgtccccac gaagggggg ctg 1920 ProSerGly ProSerCys SerGlyAsp CysProHis GluGlyGly Leu aagcataat gggcaggtg tggattctg agagaagac aggtgttca gtc 1968 LysHisAsn GlyGlnVal TrpIleLeu ArgGluAsp ArgCysSer Val tgt tcc tgt aag gat ggg aag ata ttc tgc cgg cgg aca get tgt gat 2016 Cys Ser Cys Lys Asp Gly Lys Ile Phe Cys Arg Arg Thr Ala Cys Asp tgc cag aat cca aat gtt gac ctt ttc tgc tgc cca gag tgt gac acc 2064 CysGlnAsn ProAsnVal AspLeuPhe CysCysPro GluCysAsp Thr agggtcact agccaatgt ttagatcaa agcggacag aagctctat cga 2112 ArgValThr SerGlnCys LeuAspGln SerGlyGln LysLeuTyr Arg agtggagac aactggacc caoagctgc cagcagtgc cgatgtctg gaa 2160 SerGlyAsp AsnTrpThr HisSerCys GlnGlnCys ArgCysLeu Glu ggagaggca gactgctgg cctetaget tgccctagt ttgagctgt gaa 2208 GlyGluAla AspCysTrp ProLeuAla CysProSer LeuSerCys Glu tacacagcc atctttgaa ggagagtgt tgtCCCCg'Ctgtgtcagt gac 2256 TyrThrAla IlePheGlu GlyGluCys CysProArg CysValSer Asp ccctgcctg getgataat attgcctat gacatcaga aaaacttgc ctg 2304 ProCysLeu AlaAspAsn IleAlaTyr AspIleArg LysThrCys Leu gacagctct ggtatttcg aggctgagc ggcgcagtg tggacaatg get 2352 AspSerSer GlyIleSer ArgLeuSer GlyAlaVal TrpThrMet Ala ggatctccc tgtacaacc tgtcaatgc aagaatggg agagtctgc tgc 2400 GlySerPro CysThrThr CysGlnCys LysAsnGly ArgValCys Cys tctgtggat ctggtgtgt cttgagaat aactga 2433 SerValAsp LeuValCys LeuGluAsn Asn <210>

<211> 91 <212> RT
P

<213> lus Mus muscu <400> 6 Met Pro Met Asp Val Ile Leu Val Leu Trp Phe Cys Val Cys Thr Ala Gln Asp Ser Gly Gly Leu Trp Asp Gly Pro Pro Ser Asp Gly His His His Thr Pro Cys Glu His Gln Pro Trp Ala Ser Leu Arg Trp Val Asp Tyr Thr Met Pro Val Arg His Phe Cys Phe Lys Met Tyr Arg Glu Arg Ser Thr Gln Pro Leu Met Val Arg Ser Ser Ser Tyr Ser Gly Ile Arg 65 70 . 75 80 Val Ser Leu Pro Phe Trp Leu Gln Cys Ser Arg Ser Arg Pro Pro Gln g5 90 95 Gly Tyr Cys Arg Ser Gly Ser Trp Asn Thr Ala Ile Leu Asn Trp Arg Ala Val Ala Gln Glu Lys Arg Tyr Ala Ile Ile Thr Ser Met Ala Ala Ser Pro Gly Leu Arg Pro Phe Pro Thr Ala Trp Pro Met Asp Ser Gly Thr Arg Ser Arg Cys Leu Ala Pro Leu Thr Ser Tyr Ser Met Ser Thr Ala Ile Gly Phe Met Ser Val Ile Leu Arg Arg Pro Thr Phe Leu Gln Glu Ala Ile Tyr Gly Leu Gly Asn Val Ile Lys Ser Met Ala Phe Ser Lys Glu Ser Ser Lys Met Ala Arg Ser Ser Ser Cys Arg Thr Ala Ser Ser His Ser Ala Pro Thr Ile Ala Leu Ala Gln His Ala Val Ile Ser Ala Trp Phe Lys Glu Trp Ile Cys Lys Ser Phe Trp Pro Arg Leu Gln Asn Ile Met Gln Arg Arg Asp Leu Val Asn Trp Lys Ile Ala Thr Val Arg Arg Pro Ala Lys Val Gly Cys Ser Thr Gly Thr Lys Thr Pro Gly Met Val Thr Thr Ala Gly Thr Ala His Ala Lys Val Val Leu Trp Ser Ala Glu Gly Cys Pro Val Pro His Ser Thr Val Pro Gln Thr His Phe Leu Cys Ile Phe Leu Ala Asn Val Val Lys Phe Ala Asp Gln Asn Val Ser Met Glu Glu Lys Phe Leu Leu Arg Ala Ser Gly Phe Pro Arg Pro Ala Gly Asn Val Glu Val Glu Ser Trp Lys Ser Gln Lys Leu Ala Leu Leu Thr Ala Gln Arg Arg Ile Ile Phe Phe Arg Arg Thr Ser Ala Gly Val Cys Arg Gly His Asn Phe Cys Ala Glu Ala Pro Lys Cys Gly Glu Asn Ser Glu Cys Lys Asn Trp Asn Thr Lys Ala Thr Cys Glu Cys Lys Asn Gly Tyr Ile Ser Val Gln Gly Asn Ser Ala Tyr Cys Glu Asp Ile Asp Glu Cys Ala Ala Lys Met His Tyr Cys His Ala Asn Thr Val Cys Val Asn Leu Pro Gly Leu Tyr Arg Cys Asp Cys Ile Pro Gly Tyr Tle Arg Val Asp Asp Phe Ser Cys Thr Glu His Asp Asp Cys Gly Ser Gly Gln His Asn Cys Asp Lys Asn Ala Ile Cys Thr Asn Thr Val Gln Gly His Ser Cys Thr Cys Gln Pro Gly Tyr Val Gly Asn Gly Thr Val Cys Lys Ala Phe Cys Glu Glu Gly Cys Arg Tyr Gly Gly Thr Cys Val Ala Pro Asn Lys Cys Val Cys Pro Ser Gly Phe Thr Gly Ser His Cys Glu Lys Asp Ile Asp Glu Cys Ala Glu Gly Phe Val Glu Cys His Asn His Ser Arg Cys Val Asn Leu Pro Gly Trp Tyr His Cys Glu Cys Arg Ser Gly Phe His Asp Asp Gly Thr Tyr Ser Leu Ser Gly Glu Ser Cys Ile Asp Ile Asp Glu Cys Ala Leu Arg Thr His Thr Cys Trp Asn Asp Ser Ala Cys Ile Asn Leu Ala Gly Gly Phe Asp Cys Leu Cys Pro Ser Gly Pro Ser Cys Ser Gly Asp Cys Pro His Glu Gly Gly Leu Lys His Asn Gly Gln Val Trp Ile Leu Arg Glu Asp Arg Cys Ser Val Cys Ser Cys Lys Asp Gly Lys Ile Phe Cys Arg Arg Thr Ala Cys Asp Cys Gln Asn Pro Asn Val Asp Leu Phe Cys Cys Pro Glu Cys Asp Thr Arg Val Thr Ser Gln Cys Leu Asp Gln Ser Gly Gln Lys Leu Tyr Arg Ser Gly Asp Asn Trp Thr His Ser Cys Gln Gln Cys Arg Cys Leu Glu Gly Glu Ala Asp Cys Trp Pro Leu Ala Cys Pro Ser Leu Ser Cys Glu Tyr Thr Ala Ile Phe Glu Gly Glu Cys Cys Pro Arg Cys Val Ser Asp Pro Cys Leu Ala Asp Asn Ile Ala Tyr Asp Ile Arg Lys Thr Cys Leu Asp Ser Ser Gly Ile Ser Arg Leu Ser Gly Ala Val Trp Thr Met Ala Gly Ser Pro Cys Thr Thr Cys Gln Cys Lys Asn Gly Arg Val Cys Cys Ser Val Asp Leu Val Cys Leu Glu Asn Asn <210> 7 <211> 2451 <212> DNA
<213> Homo sapiens <220>
<221> CDS
<222> (1)...(2451) <400> 7 atg gag tct cgg gtc tta ctg aga aca ttc tgt ttg atc ttc ggt ctc 48 Met Glu Ser Arg Val Leu Leu Arg Thr Phe Cys Leu Ile Phe Gly Leu gga gca gtt tgg ggg ctt ggt gtg gac cct tcc cta cag att gac gtc 96 Gly Ala Val Trp Gly Leu Gly Val Asp Pro Ser Leu Gln Ile Asp Val tta tta cttggg tccacg ggagtgcgt cag 144 aca gaa gag acc gtc gag Leu Glu LeuGlyGlu SerThr GlyValArg Gln Thr Thr Val Glu Leu ccgggg ctgcataat gggacgaaa gcctttctc tttcaagat actccc 192 ProGly LeuHisAsn GlyThrLys AlaPheLeu PheGlnAsp ThrPro agaagc ataaaagca tccactget acagetgaa cagtttttt cagaag 240 ArgSer IleLysAla SerThrAla ThrAlaGlu GlnPhePhe GlnLys ctgaga aataaacat gaatttact attttggtg accctaaaa cagacc 288 LeuArg AsnLysHis GluPheThr IleLeuVal ThrLeuLys GlnThr cactta aattcagga gttattctc tcaattcac cacttggat cacagg 336 HisLeu AsnSerGly ValIleLeu SerIleHis HisLeuAsp HisArg tacctg gaactggaa agtagtggc catcggaat gaagtcaga ctgcat 384 TyrLeu GluLeuGlu SerSerGly HisArgAsn GluValArg LeuHis taccgc tcaggcagt Ca.CCgCCCt cacacagaa gtgtttcct tacatt 432 TyrArg SerGlySer HisArgPro HisThrGlu ValPhePro TyrIle ttgget gatgacaag tggcacaag ctctcctta gccatcagt gettcc 480 LeuAla AspAspLys TrpHisLys LeuSerLeu AlaIleSer AlaSer catttg attttacac attgactgc aataaaatt tatgaaagg gtagta 528 HisLeu IleLeuHis IleAspCys AsnLysIle TyrGluArg ValVal gaaaag ccctccaca gacttgcct ctaggcaca acattttgg ctagga 576 GluLys ProSerThr AspLeuPro LeuGlyThr ThrPheTrp LeuGly cagaga aataatgcg catggatat tttaagggt ataatgcaa gatgtc 624 GlnArg AsnAsnAla HisGlyTyr PheLysGly IleMetGln AspVal caatta cttgtcatg ccccaggga tttattget cagtgccca gatctt 672 GlnLeu LeuValMet ProGlnGly PheIleAla GlnCysPro AspLeu aatcgc acctgtcca acttgcaat gacttccat ggacttgtg cagaaa 720 AsnArg ThrCysPro ThrCysAsn AspPheHis Gly GlnLys Leu Val atc atg gag cta cag gat att tta gcc aaa aca tca gcc aag ctg tct 768 Ile Met Glu Leu Gln Asp Ile Leu Ala Lys Thr Ser Ala Lys Leu Ser cga get gaa cag cga atg aat aga ttg gat cag tgc tat tgt gaa agg 816 Arg Ala Glu Gln Arg Met Asn Arg Leu Asp Gln Cys Tyr Cys Glu Arg acttgcacc atgaagggaacc acctac cgagaattt gagtcctgg ata 864 ThrCysThr MetLysGlyThr ThrTyr ArgGluPhe GluSerTrp Ile gacggctgt aagaactgcaca tgcctg aatggaacc atccagtgt gaa 912 AspGlyCys LysAsnCysThr CysLeu AsnGlyThr IleGlnCys Glu actctaatc tgcccaaatcct gactgc ccacttaag tcggetctt gcg 960 ThrLeuIle CysProAsnPro AspCys ProLeuLys SerAlaLeu Ala tatgtggat ggcaaatgctgt aaggaa tgcaaatcg atatgccaa ttt 1008 TyrValAsp GlyLysCysCys LysGlu CysLysSer IleCysGln Phe caaggacga acctacttt gaaggagaa agaaataca gtctattcc tct 1056 GlnGlyArg ThrTyrPhe GluGlyGlu ArgAsnThr ValTyrSer Ser tctggagta tgtgttctc tatgagtgc aaggaccag accatgaaa ctt 1104 SerGlyVal CysValLeu TyrGluCys LysAspGln ThrMetLys Leu gttgagagt tcaggctgt ccagetttg gattgtcca gagtctcat cag 1152 ValGluSer SerGlyCys ProAlaLeu AspCysPro GluSerHis Gln ataaccttg tctcacagc tgttgcaaa gtttgtaaa ggttatgac ttt 1200 21eThrLeu SerHisSer CysCysLys ValCysLys GlyTyrAsp Phe .

tgttctgaa aggcataac tgcatggag aattccatc tgcagaaat ctg 1248 CysSerGlu ArgHisAsn CysMetGlu AsnSerIle CysArgAsn Leu aatgacagg getgtttgt agctgtcga gatggtttt agggetctt cga 1296 AsnAspArg AlaValCys SerCysArg AspGlyPhe ArgAlaLeu Arg gaggataat gcctactgt gaagacatc gatgagtgt getgaaggg cgc 1344 GluAspAsn AlaTyrCys GluAspIle AspGluCys AlaGluGly Arg _ 22 _ cat tac tgt cgt gaa aat aca atg tgt gtc aac.acc ccg ggt tct ttt 1392 His Tyr Cys Arg Glu Asn Thr Met Cys Val Asn Thr Pro Gly Ser Phe atg tgc atc tgc aaa act gga tac atc aga att gat gat tat tca tgt 1440 Met Cys Ile Cys Lys Thr Gly Tyr Ile Arg Ile Asp Asp Tyr Ser Cys aca gaa cat gat gag tgt atc aca aat cag cac aac tgt gat gaa aat 1488 Thr Glu His Asp Glu Cys Ile Thr Asn Gln His Asn Cys Asp Glu Asn get tta tgc ttc aac act gtt gga gga cac aac tgt gtt tgc aag ccg 1536 Ala Leu Cys Phe Asn Thr Val Gly Gly His Asn Cys Val Cys Lys Pro ggc tat aca ggg aat gga acg aca tgc aaa gca ttt tgc aaa gat ggc 1584 Gly Tyr Thr Gly Asn Gly Thr Thr Cys Lys Ala Phe Cys Lys Asp Gly tgt agg aat gga gga gcc tgt att gcc get aat gtg tgt gcc tgc cca 1632 Cys Arg Asn Gly Gly Ala Cys Ile Ala Ala Asn Val Cys Ala Cys Pro caa ggc ttc act gga ccc agc tgt gaa acg gac att gat gaa tgc tct 1680 Gln Gly Phe Thr Gly Pro Ser Cys Glu Thr Asp Ile Asp Glu Cys Ser 545 550 555 5'60 gat ggt ttt gtt caa tgt gac agt cgt get aat tgc att aac ctg cct 1728 Asp Gly Phe Val Gln Cys Asp Ser Arg Ala Asn Cys Ile Asn Leu Pro gga tgg tac cac tgt gag tgc aga gat ggc tac cat gac aat ggg atg 1776 Gly Trp Tyr His Cys Glu Cys Arg Asp Gly Tyr His Asp Asn Gly Met ttt tca cca agt gga gaa tcg tgt gaa gat att gat gag tgt ggg acc 1824 Phe Ser Pro Ser Gly Glu Ser Cys Glu Asp Ile Asp Glu Cys Gly Thr ggg agg cac agc tgt gcc aat gat acc att tgc ttc aat ttg gat ggc 1872 Gly Arg His Ser Cys Ala Asn Asp Thr Ile Cys Phe Asn Leu Asp Gly gga tat gat tgt cga tgt cct cat gga aag aat tgc aca ggg gac tgc 1920 Gly Tyr Asp Cys Arg Cys Pro His Gly Lys Asn Cys Thr Gly Asp Cys atc cat gat gga aaa gtt aag cac aat ggt cag att tgg gtg ttg gaa 1968 Ile His Asp Gly Lys Val Lys His Asn Gly Gln Ile Trp Val Leu Glu aat gac agg tgc tct gtg tgc tca tgt cag aat gga ttc gtt atg tgt 2016 Asn Asp Arg Cys Ser Val Cys Ser Cys Gln Asn Gly Phe Val Met Cys cga cgg atg gtc tgt gac tgt gag aat ccc aca gtt gat ctt ttt tgc 2064 Arg Arg Met Val Cys Asp Cys Glu Asn Pro Thr Val Asp Leu Phe Cys tgc cct gaa tgt gac cca agg ctt agt agt cag tgc ctc cat caa aat 2112 Cys Pro Glu Cys Asp Pro Arg Leu Ser Ser Gln Cys Leu His Gln Asn ggg gaa act ttg tat aac agt ggt gac acc tgg gtc cag aat tgt caa 2160 Gly Glu Thr Leu Tyr Asn Ser Gly Asp Thr Trp Val Gln Asn Cys Gln cag tgc cgc tgc ttg caa ggg gaa gtt gat tgt tgg ccc ctg cct tgc 2208 Gln Cys Arg Cys Leu Gln Gly Glu Val Asp Cys Trp Pro Leu Pro Cys cca gat gtg gag tgt gaa ttc agc att ctc cca gag aat gag tgc tgc 2256 Pro Asp Val Glu Cys Glu Phe Ser Ile Leu Pro Glu Asn Glu Cys Cys ccg cgc tgt gtc aca gac cct tgc cag get gac acc atc cgc aat gac 2304 Pro Arg Cys Val Thr Asp Pro Cys Gln Ala Asp Thr Ile Arg Asn Asp atc acc aag act tgc ctg gac gaa atg aat gtg gtt cgc ttc acc ggg 2352 Ile Thr Lys Thr Cys Leu Asp Glu Met Asn Val Val Arg Phe Thr Gly tcc tct tgg atc aaa cat ggc act gag tgt act ctc tgc cag tgc aag 2400 Ser Ser Trp Ile Lys His Gly Thr Glu Cys Thr Leu Cys Gln Cys Lys aat ggc cac atc tgt tgc tca gtg gat cca cag tgc ctt cag gaa ctg 2448 Asn Gly His Ile Cys Cys Ser Val Asp Pro Gln Cys Leu Gln Glu Leu tga 2451 <210> 8 <211> 816 <212> PRT
<213> Homo Sapiens <400> 8 Met Glu Ser Arg Val Leu Leu Arg Thr Phe Cys Leu Ile Phe Gly Leu Gly Ala Val Trp Gly Leu Gly Val Asp Pro Ser Leu Gln Ile Asp Val Leu Thr Glu Leu Glu Leu Gly Glu Ser Thr Thr Gly Val Arg Gln Val Pro Gly Leu His Asn Gly Thr Lys Ala Phe Leu Phe Gln Asp Thr Pro Arg Ser Ile Lys Ala Ser Thr Ala Thr Ala Glu Gln Phe Phe Gln Lys Leu Arg Asn Lys His Glu Phe Thr Ile Leu Val Thr Leu Lys Gln Thr His Leu Asn Ser Gly Val Ile Leu Ser Ile His His Leu Asp His Arg Tyr Leu Glu Leu Glu Ser Ser Gly His Arg Asn Glu Val Arg Leu His Tyr Arg Ser Gly Ser His Arg Pro His Thr Glu Val Phe Pro Tyr Ile Leu Ala Asp Asp Lys Trp His Lys Leu Ser Leu Ala Ile Ser Ala Ser His Leu Ile Leu His Ile Asp Cys Asn Lys Ile Tyr Glu Arg Val Val Glu Lys Pro Ser Thr Asp Leu Pro Leu Gly Thr Thr Phe Trp Leu Gly 180 ' 185 190 Gln Arg Asn Asn Ala His Gly Tyr Phe Lys Gly Ile Met Gln Asp Val Gln Leu Leu Val Met Pro Gln Gly Phe Ile Ala Gln Cys Pro Asp Leu Asn Arg Thr Cys Pro Thr Cys Asn Asp Phe His Gly Leu Val Gln Lys Ile Met Glu Leu Gln Asp Ile Leu Ala Lys Thr Ser Ala Lys Leu Ser Arg Ala Glu Gln Arg Met Asn Arg Leu Asp Gln Cys Tyr Cys Glu Arg Thr Cys Thr Met Lys Gly Thr Thr Tyr Arg Glu Phe Glu Ser Trp Ile Asp Gly Cys Lys Asn Cys Thr Cys Leu Asn Gly Thr Ile Gln Cys Glu Thr Leu Ile Cys Pro Asn Pro Asp Cys Pro Leu Lys Ser Ala Leu Ala Tyr Val Asp Gly Lys Cys Cys Lys Glu Cys Lys Ser Ile Cys Gln Phe Gln Gly Arg Thr Tyr Phe Glu Gly Glu Arg Asn Thr Val Tyr Ser Ser Ser Gly Val Cys Val Leu Tyr Glu Cys Lys Asp Gln Thr Met Lys Leu Val Glu Ser Ser Gly Cys Pro Ala Leu Asp Cys Pro Glu Ser His Gln Ile Thr Leu Ser His Ser Cys Cys Lys Val Cys Lys Gly Tyr Asp Phe Cys Ser Glu Arg His Asn Cys Met Glu Asn Ser Ile Cys Arg Asn Leu Asn Asp Arg Ala Val Cys Ser Cys Arg Asp Gly Phe Arg Ala Leu Arg Glu Asp Asn Ala Tyr Cys Glu Asp Ile Asp Glu Cys Ala Glu Gly Arg His Tyr Cys Arg Glu Asn Thr Met Cys Val Asn Thr Pro Gly Ser Phe Met Cys Ile Cys Lys Thr Gly Tyr Ile Arg Ile Asp Asp Tyr Ser Cys Thr Glu His Asp Glu Cys Ile Thr Asn Gln His Asn Cys Asp Glu Asn Ala Leu Cys Phe Asn Thr Val Gly Gly His Asn Cys Val Cys Lys Pro Gly Tyr Thr Gly Asn Gly Thr Thr Cys Lys Ala Phe Cys Lys Asp Gly Cys Arg Asn Gly Gly Ala Cys Ile Ala Ala Asn Val Cys Ala Cys Pro Gln Gly Phe Thr Gly Pro Ser Cys Glu Thr Asp Ile Asp Glu Cys Ser Asp Gly Phe Val Gln Cys Asp Ser Arg Ala Asn Cys Ile Asn Leu Pro Gly Trp Tyr His Cys Glu Cys Arg Asp Gly Tyr His Asp Asn Gly Met Phe Ser Pro Ser Gly Glu Ser Cys Glu Asp Ile Asp Glu Cys Gly Thr Gly Arg His Ser Cys Ala Asn Asp Thr Ile Cys Phe Asn Leu Asp Gly Gly Tyr Asp Cys Arg Cys Pro His Gly Lys Asn Cys Thr Gly Asp Cys Ile His Asp Gly Lys Val Lys His Asn Gly Gln Ile Trp Val Leu Glu Asn Asp Arg Cys Ser Val Cys Ser Cys Gln Asn Gly Phe Val Met Cys Arg Arg Met Val Cys Asp Cys Glu Asn Pro Thr Val Asp Leu Phe Cys Cys Pro Glu Cys Asp Pro Arg Leu Ser Ser Gln Cys Leu His Gln Asn Gly Glu Thr Leu Tyr Asn Ser Gly Asp Thr Trp Val Gln Asn Cys Gln Gln Cys Arg Cys Leu Gln Gly Glu Val Asp Cys Trp Pro Leu Pro Cys Pro Asp Val Glu Cys Glu Phe Ser Ile Leu Pro Glu Asn Glu Cys Cys Pro Arg Cys Val Thr Asp Pro Cys Gln Ala Asp Thr Ile Arg Asn Asp Ile Thr Lys Thr Cys Leu Asp Glu Met Asn Val Val Arg Phe Thr Gly Ser Ser Trp Ile Lys His Gly Thr Glu Cys Thr Leu Cys Gln Cys Lys Asn Gly His Ile Cys Cys Ser Val Asp Pro Gln Cys.Leu Gln Glu Leu <210> 9 <211> 2451 <212> DNA
<213> Rattus norvegicus <220>

<221> CDS
<222> (1)...(2451) <400> 9 atg gaa tcc cgg gta tta ctg aga acg ttc tgc gtg atc ctc ggg ctc 48 Met Glu Ser Arg Val Leu Leu Arg Thr Phe Cys Val Ile Leu Gly Leu gaa gcg gtt tgg gga ctt ggt gtg gac ccc tcc cta cag att gac gtc 96 Glu Ala Val Trp Gly Leu Gly Val Asp Pro Ser Leu Gln Ile Asp Val tta tca gag tta gaa ctt ggg gag tcc aca get gga gtg cgc caa gtc 144 Leu Ser Glu Leu Glu Leu Gly Glu Ser Thr Ala Gly Val Arg Gln Val ccagga ctgcataat gggacgaaagcc ttCCtCttc caagattcc ccc 192 ProGly LeuHisAsn GlyThrLysAla PheLeuPhe GlnAspSer Pro agaagc ataaaagca cccattgetaca getgagcgg tttttccag aag 240 ArgSer IleLysAla ProIleAlaThr AlaGluArg PhePheGln Lys ctgagg aataaacac gagttcacaatt ctggtgacc ctgaaacag atc 288 LeuArg AsnLysHis GluPheThrIle LeuValThr LeuLysGln Ile cactta aattcggga gtcattctctcc atccaccac ttggatcac agg 336 HisLeu AsnSerGly ValIleLeuSer IleHisHis LeuAspHis Arg tacctg gaactggaa agcagcggccac cggaatgag atcagactg cat 384 TyrLeu GluLeuGlu SerSerGlyHis ArgAsnGlu IleArgLeu His taccgc tctggaact caccgcccgcac acggaagtg tttccttat.att 432 TyrArg SerGlyThr HisArgProHis ThrGluVal PheProTyr Ile ttgget gatgccaag tggcacaagctc tccttagcc ttcagtgcc tcc 480 LeuAla AspAlaLys TrpHisLysLeu SerLeuAla PheSerAla Ser cactta attttacac atcgactgcaac aagatctat gaacgagtg gtg 528 HisLeu IleLeuHis IleAspCysAsn LysIleTyr GluArgVal Val gaaatg ccttctaca gacttgcctctg ggcaccaca ttttggttg gga 576 GluMet ProSerThr AspLeuProLeu GlyThrThr PheTrpLeu Gly cagaga aataacgca cacgggtatttt aagggaata atgcaagat gtg 624 Gln Arg Asn Asn Ala His Gly Tyr Phe Lys Gly I1e Met Gln Asp Val caa tta ctt gtc atg ccc cag ggg ttc atc get cag tgc ccg gat ctt 672 Gln Leu Leu Val Met Pro Gln Gly Phe Ile Ala Gln Cys Pro Asp Leu 210 215 . 220 aat cga acc tgt cca aca tgc aac gac ttc cat ggg ctt gtg cag aaa 720 Asn Arg Thr Cys Pro Thr Cys Asn Asp Phe His Gly Leu Val Gln Lys atc atg gag ctg cag gac att tta tcg aag acg tca gcc aag ttg tct 768 Ile Met Glu Leu Gln Asp Ile Leu Ser Lys Thr Ser Ala Lys Leu Ser aga get gaa caa cga atg aac agg ctg gat cag tgc tac tgt gag cgg 816 Arg Ala Glu Gln Arg Met Asn Arg Leu Asp Gln Cys Tyr Cys Glu Arg acg tgc acc atg aag gga gcc acc tac cgg gag ttc gag tcc tgg aca 864 Thr Cys Thr Met Lys Gly Ala Thr Tyr Arg Glu Phe Glu Ser Trp Thr gac ggc tgc aag aac tgc aca tgc ttg aat ggg acc atc cag tgc gag 912 Asp Gly Cys Lys Asn Cys Thr Cys Leu Asn Gly Thr Ile Gln Cys Glu act ctg gtc tgc cct get ccc gac tgc ccg get aaa tcg get cca gcg 960 Thr Leu Val Cys Pro Ala Pro Asp Cys Pro Ala Lys Ser Ala Pro Ala tac gtg gat ggc aag tgc tgt aag gag tgc aag tcc acc tgc cag ttc 1008 Tyr Val Asp Gly Lys Cys Cys Lys Glu Cys Lys Ser Thr Cys Gln Phe cag ggg cgg agc tac ttt gag gga gaa agg agc aca gtc ttc tca get 1056 Gln Gly Arg Ser Tyr Phe Glu Gly Glu Arg Ser Thr Val Phe Ser Ala tcc gga atg tgc gtc ttg tat gaa tgc aag gat cag acc atg aag ctt 1104 Ser Gly Met Cys Val Leu Tyr Glu Cys Lys Asp Gln Thr Met Lys Leu gtt gag aac gcc ggc tgc ccg get tta gat tgc ccc gag tct cat cag 1152 Val Glu Asn Ala Gly Cys Pro Ala Leu Asp Cys Pro Glu Ser His Gln atc gcc ttg tct cac agc tgc tgc aag gtt tgc aaa ggt tat gac ttc 1200 Ile Ala Leu Ser His Ser Cys Cys Lys Val Cys Lys Gly Tyr Asp Phe tgt tct gag aag cat aca tgc atg gag aac tca gtc tgc agg aac ctg 1248 Cys Ser Glu Lys His Thr Cys Met Glu Asn Ser Val Cys Arg Asn Leu _ 28 _ aac gac agg gca gtg tgc agc tgc cgg gat ggt ttc cgg gcc ctc cgg 1296 Asn Asp Arg Ala Val Cys Ser Cys Arg Asp Gly Phe Arg Ala Leu Arg gag gac aat gcc tac tgt gaa gac att gac gag tgt gca gag ggg cgc 1344 Glu Asp Asn Ala Tyr Cys Glu Asp Ile Asp Glu Cys Ala Glu Gly Arg cat tac tgc cgt gag aac acc atg tgt gtg aac aca ccg ggc tct ttc 1392 His Tyr Cys Arg Glu Asn Thr Met Cys Val Asn Thr Pro Gly Ser Phe ctg tgt atc tgc caa aca ggg tac atc aga atc gac gat tac tcg tgt 1440 Leu Cys Ile Cys Gln Thr Gly Tyr Ile Arg Ile Asp Asp Tyr Ser Cys acg gaa cat gac gag tgc ctc aca aac cag cac aac tgt gac gag aac 1488 Thr Glu His Asp Glu Cys Leu Thr Asn Gln His Asn Cys Asp Glu Asn get ttg tgc ttt aac acc gtt gga ggt cac aac tgc gtc tgc aag cct 1536 Ala Leu Cys Phe Asn Thr Val Gly Gly His Asn Cys Val Cys Lys Pro ggg tac act ggg aat gga acc acg tgc aaa get ttc tgc aaa gac ggc 1584 Gly Tyr Thr Gly Asn Gly Thr Thr Cys Lys Ala Phe Cys Lys Asp Gly tgc aaa aac gga ggt gcc tgc att get gcc aat gtc tgt get tgc cca 1632 Cys Lys Asn Gly Gly Ala Cys Ile Ala Ala Asn Val Cys Ala Cys Pro caa ggc ttc acc gga ccc agc tgt gag aca gac att gat gag tgc tct 1680 Gln Gly Phe Thr Gly Pro Ser Cys Glu Thr Asp Ile Asp Glu Cys Ser gag ggc ttt gtt cag tgt gac agc cgt gcc aac tgc att aac ctg cct 1728 Glu Gly Phe Val Gln Cys Asp Ser Arg Ala Asn Cys Ile Asn Leu Pro ggg tgg tac cac tgt gag tgc aga gat ggc tac cat gac aat ggg atg 1776 Gly Trp Tyr His Cys Glu Cys Arg Asp Gly Tyr His Asp Asn Gly Met ttt gcg cca ggt gga gaa tec tgt gaa gat att gat gaa tgt ggg act 1824 Phe Ala Pro Gly Gly Glu Ser Cys Glu Asp Ile Asp Glu Cys Gly Thr ggg agg cac agc tgt gcc aat gac acc att tgc ttc aac ttg gac ggt 1872 Gly Arg His Ser Cys Ala Asn Asp Thr Ile Cys Phe Asn Leu Asp Gly ggc tac gat tgc cgg tgt ccc cat gga aag aac tgc aca ggg gac tgc 1920 Gly Tyr Asp Cys Arg Cys Pro His Gly Lys Asn Cys Thr Gly Asp Cys gtg cac gac ggg aaa gtc aaa cac aac ggc cag atc tgg gtg ctg gag 1968 Val His Asp Gly Lys Val Lys His Asn Gly Gln Ile Trp Val Leu Glu aac gac agg tgc tct gtg tgt tcc tgc cag act gga ttt gtt atg tgc 2016 Asn Asp Arg Cys Ser Val Cys Ser Cys Gln Thr Gly Phe Val Met Cys caa cgg atg gtc tgt gac tgc gaa aac ccc aca gtt gac etc tcc tgc 2064 Gln Arg Met Val Cys Asp Cys Glu Asn Pro Thr Val Asp Leu Ser Cys tgc cct gag tgc gac cca agg ctg agc agc cag tgc ctg cat caa aac 2112 Cys Pro Glu Cys Asp Pro Arg Leu Ser Ser Gln Cys Leu His Gln Asn ggg gaa acc gtg tac aac agc ggt gac acc tgg gcc cag gat tgc cgt 2160 Gly Glu Thr Val Tyr Asn Ser Gly Asp Thr Trp Ala Gln Asp Cys Arg cag tgc cgc tgc ttg caa gaa gaa gtt gac tgc tgg CCC ctg get tgc 2208 Gln Cys Arg Cys Leu Gln Glu Glu Val Asp Cys Trp Pro Leu Ala Cys cca gag gta gag tgt gaa ttt agt gtc ctt cct gag aac gag tgc tgc 2256 Pro Glu Val Glu Cys Glu Phe Ser Val Leu Pro Glu Asn Glu Cys Cys cca cgc tgt gtc acc gat cct tgt cag get gac acc atc cgc aat gac 2304 Pro Arg Cys Val Thr Asp Pro Cys Gln Ala Asp Thr Ile Arg Asn Asp atc acc aaa acc tgc ctg gac gag atg aac gtg gtt cgc ttc act ggg 2352 Ile Thr Lys Thr Cys Leu Asp Glu Met Asn Val Val Arg Phe Thr Gly tct tcc tgg atc aag cac ggc acg gag tgc acc ctc tgc cag tgc aag 2400 Ser Ser Trp Ile Lys His Gly Thr Glu Cys Thr Leu Cys Gln Cys Lys aac ggc cac gtg tgc tgc tca gtg gac cca cag tgc ctc cag gag ctg 2448 Asn Gly His Val Cys Cys Ser Val Asp Pro Gln Cys Leu Gln Glu Leu tga 2451 <210> 10 <211> 816 <212> PRT
<213> Rattus norvegicus <400> 10 Met Glu Ser Arg Val Leu Leu Arg Thr Phe Cys Val Ile Leu Gly Leu Glu Ala Val Trp Gly Leu Gly Val Asp Pro Ser Leu Gln Ile Asp Val Leu Ser Glu Leu Glu Leu Gly Glu Ser Thr Ala Gly Val Arg Gln Val Pro Gly Leu His Asn Gly Thr Lys Ala Phe Leu Phe Gln Asp Ser Pro Arg Ser Ile Lys Ala Pro Ile Ala Thr Ala Glu Arg Phe Phe Gln Lys Leu Arg Asn Lys His Glu Phe Thr Ile Leu Val Thr Leu Lys Gln Ile His Leu Asn Ser Gly Val Ile Leu Ser Ile His His Leu Asp His Arg Tyr Leu Glu Leu Glu Ser Ser Gly His Arg Asn Glu Ile Arg Leu His Tyr Arg Ser Gly Thr His Arg Pro His Thr Glu Val Phe Pro Tyr Ile Leu Ala Asp Ala Lys Trp His Lys Leu Ser Leu Ala Phe Ser Ala Ser His Leu Ile Leu His Ile Asp Cys Asn Lys Ile Tyr Glu Arg Val Val Glu Met Pro Ser Thr Asp Leu Pro Leu Gly Thr Thr Phe Trp Leu Gly Gln Arg Asn Asn Ala His Gly Tyr Phe Lys Gly Ile Met Gln Asp Val Gln Leu Leu Val Met Pro Gln Gly Phe Ile Ala Gln Cys Pro Asp Leu Asn Arg Thr Cys Pro Thr Cys Asn Asp Phe His Gly Leu Val Gln Lys Ile Met Glu Leu Gln Asp Ile Leu Ser Lys Thr Ser Ala Lys Leu Ser Arg Ala Glu Gln Arg Met Asn Arg Leu Asp Gln Cys Tyr Cys Glu Arg Thr Cys Thr Met Lys Gly Ala Thr Tyr Arg Glu Phe Glu Ser Trp Thr Asp Gly Cys Lys Asn Cys Thr Cys Leu Asn Gly Thr Ile Gln Cys Glu Thr Leu Val Cys Pro Ala Pro Asp Cys Pro Ala Lys Ser Ala Pro Ala Tyr Val Asp Gly Lys Cys Cys Lys Glu Cys Lys Ser Thr Cys Gln Phe Gln Gly Arg Ser Tyr Phe Glu Gly Glu Arg Ser Thr Val Phe Ser Ala Ser Gly Met Cys Val Leu Tyr Glu Cys Lys Asp Gln Thr Met Lys Leu Val Glu Asn Ala Gly Cys Pro Ala Leu Asp Cys Pro Glu Ser His Gln Ile Ala Leu Ser His Ser Cys Cys Lys Val Cys Lys Gly Tyr Asp Phe Cys Ser Glu Lys His Thr Cys Met Glu Asn Ser Val Cys Arg Asn Leu Asn Asp Arg Ala Val Cys Ser Cys Arg Asp Gly Phe Arg Ala Leu Arg Glu Asp Asn Ala Tyr Cys Glu Asp Ile Asp Glu Cys Ala Glu Gly Arg His Tyr Cys Arg Glu Asn Thr Met Cys Val Asn Thr Pro Gly Ser Phe Leu Cys Ile Cys Gln Thr Gly Tyr Ile Arg Ile Asp Asp Tyr Ser Cys Thr Glu His Asp Glu Cys Leu Thr Asn Gln His Asn Cys Asp Glu Asn Ala Leu Cys Phe Asn Thr Val Gly Gly His Asn Cys Val Cys Lys Pro Gly Tyr Thr Gly Asn Gly Thr Thr Cys Lys Ala Phe Cys Lys Asp Gly ~Cys Lys Asn Gly Gly Ala Cys Ile Ala Ala Asn Val Cys Ala Cys Pro Gln Gly Phe Thr Gly Pro Ser Cys Glu Thr Asp Ile Asp Glu Cys Ser Glu Gly Phe Val Gln Cys Asp Ser Arg Ala Asn Cys Ile Asn Leu Pro Gly Trp Tyr His Cys Glu Cys Arg Asp Gly Tyr His Asp Asn Gly Met 580. 585 590 Phe Ala Pro Gly Gly Glu Ser Cys Glu Asp Ile Asp Glu Cys Gly Thr Gly Arg His Ser Cys Ala Asn Asp Thr Ile Cys Phe Asn Leu Asp Gly Gly Tyr Asp Cys Arg Cys Pro His Gly Lys Asn Cys Thr Gly Asp Cys Val His Asp Gly Lys Val Lys His Asn Gly Gln Ile Trp Val Leu Glu Asn Asp Arg Cys Ser Val Cys Ser Cys Gln Thr Gly Phe Val Met Cys Gln Arg Met Val Cys Asp Cys Glu Asn Pro Thr Val Asp Leu Ser Cys Cys Pro Glu Cys Asp Pro Arg Leu Ser Ser Gln Cys Leu His Gln Asn Gly Glu Thr Val Tyr Asn Ser Gly Asp Thr Trp Ala Gln Asp Cys Arg Gln Cys Arg Cys Leu Gln Glu Glu Val Asp Cys Trp Pro Leu Ala Cys Pro Glu Val Glu Cys Glu Phe Ser Val Leu Pro Glu Asn Glu Cys Cys Pro Arg Cys Val Thr Asp Pro Cys Gln Ala Asp Thr Ile Arg Asn Asp Ile Thr Lys Thr Cys Leu Asp Glu Met Asn Val Val Arg Phe Thr Gly Ser Ser Trp Ile Lys His Gly Thr Glu Cys Thr Leu Cys Gln Cys Lys Asn Gly His Val Cys Cys Ser Val Asp Pro Gln Cys Leu Gln Glu Leu <210> 11 <211> 2460 <212> DNA
<213> Mus musculus <220>
<221> CDS
<222> (1)...(2460) <221> misc_feature <222> (1) . . (2460) <223> n = A,T,C or G
<400>

atgcacgcc atggaa tcccgggtgtta ctgagaacg ttctgcgtg atc 48 MetHisAla MetGlu SerArgValLeu LeuArgThr PheCysVal Ile ctcggcctt ggagcg gtttgggggctt ggtgtggac ccctcccta cag 96 LeuGlyLeu GlyAla ValTrpGlyLeu GlyValAsp ProSerLeu Gln attgacgtc ttaaca gagttagaactt ggggagtct acagatgga gtg 144 IleAspVal LeuThr GluLeuGluLeu GlyGluSer ThrAspGly Val egccaagtc ecggga ctgcataatggg acgaaagcc ttcctcttc caa 192 ArgGlnVal ProGly LeuHisAsnGly ThrLysAla PheLeuPhe Gln gagtecccc agaagc ataaaggcatcc actgetaca getgagcgg ttt 240 GluSerPro ArgSer IleLysAlaSer ThrAlaThr AlaGluArg Phe ctccagaag ctgaga aataaacacgag ttcacaatc ttggtgacc tta 288 LeuGlnLys LeuArg AsnLysHisGlu PheThrIle LeuValThr Leu aaacagatc cactta aattcgggagtt atcctctcc atccaccac ttg 336 LysGlnIle HisLeu AsnSerGlyVal IleLeuSer IleHisHis Leu gatcacagg tacetg gaactggaaagc agtggccat cggaatgag atc 384 AspHisArg TyrLeu GluLeuGluSer SerGlyHis ArgAsnGlu Ile agaetccac taccgc tctggcactcac cgcccccac acggaagtg ttt 432 ArgLeuHis TyrArg SerGlyThrHis ArgProHis ThrGluVal Phe ccttatatt ttggetgat gccaagtgg cacaagctc tccttagcc ttc 480 ProTyrIle LeuAlaAsp AlaLysTrp HisLysLeu SerLeuAla Phe agtgcctct cacttaatt ttacacatc gactgcaat aagatctat gaa 528 SerAlaSer HisLeuIle LeuHisIle AspCysAsn LysIleTyr Glu cgagtggtg gaaatgccc ttcacagac ttggetctg ggcacaaca ttt 576 ArgValVal GluMetPro PheThrAsp LeuAlaLeu GlyThrThr Phe tggttggga cagagaaat aatgcacat ggctatttt aagggaata atg 624 TrpLeuGly GlnArgAsn AsnAlaHis GlyTyrPhe LysGlyIle Met caggatgtg cacgtnctt gtcatgcct cagggcttc attgetcag tgc 672 GlnAspVal HisValLeu ValMetPro GlnGlyPhe IleAlaGln Cys ccggacctt aatcgaacc tgtccaaca tgcaacgac ttccatggg ctt 720' ProAspLeu AsnArgThr CysProThr CysAsnAsp PheHisGly Leu gtgcagaaa atcatggag ctgcaggac attttatca aagacgtca gcc 768 ValGlnLys IleMetGlu LeuGlnAsp IleLeuSer LysThrSer Ala aagctgtcc cgagetgaa caaagaatg aacaggctg gatcagtgc tac 816 LysLeuSer ArgAlaGlu GlnArgMet AsnArgLeu AspGlnCys Tyr tgtgagcgg acatgcact gtgaaggga accacctac cgagagtct gag 864 CysGluArg ThrCysThr ValLysGly ThrThrTyr ArgGluSer Glu tcctggaca gacggctgt aagaactgc acatgcttg aacgggacc atc 912 SerTrpThr AspGlyCys LysAsnCys ThrCysLeu AsnGlyThr Ile cagtgcgag actctggtc tgccctget cctgactgc cctcctaaa tcg 960 GlnCysGlu ThrLeuVal CysProAla ProAspCys ProProLys Ser gcccctgcg tatgtggat ggcaagtgc tgtaaggag tgcaaatca acc 1008 AlaProAla TyrValAsp GlyLysCys CysLysGlu CysLysSer Thr tgccagttc cagggacgg agctacttt gagggagaa aggaacacg gca 1056 CysGlnPhe GlyArg SerTyrPhe GluGlyGlu Arg Thr Ala Gln Asn tactca tcttctgga atgtgtgtctta tatgaatgc aaggatcag 1104 acc TyrSer SerSerGly MetCysValLeu TyrGluCys LysAspGln Thr atgaag cttgttgag aacattggctgc ccaccctta gattgtccc gag 1152 MetLys LeuValGlu AsnIleGlyCys ProProLeu AspCysPro Glu tctcat cagattgcc ttgtctcacagc tgctgcaag gtttgtaaa ggt 1200 SerHis GlnIleAla LeuSerHisSer CysCysLys ValCysLys Gly tatgac ttctgttct gagaagcatacc tgcatggag aactcggtc tgc 1248 TyrAsp PheCysSer GluLysHisThr CysMetGlu AsnSerVal Cys aggaac ctgaacgac agggttgtgtgc agctgcagg gatggtttt cgg 1296 ArgAsn LeuAsnAsp ArgValValCys SerCysArg AspGlyPhe Arg getctc cgagaggac aacgcctactgt gaagacatt gacgagtgt gca 1344 AlaLeu ArgGluAsp AsnAlaTyrCys GluAspIle AspGluCys Ala gaaggg cgccattac tgccgtgagaac accatgtgt gtgaataca cct 1392 GluGly ArgHisTyr CysArgGluAsn ThrMetCys ValAsnThr Pro ggttct ttcatgtgt gtctgcaaaact gggtacatc aggatcgac gat 1440 GlySer PheMetCys ValCysLysThr GlyTyrIle ArgIleAsp Asp tactca tgtacagaa catgatgagtgt ctcacaacc cagcacaat tgt 1488 TyrSer CysThrGlu HisAspGluCys LeuThrThr GlnHisAsn Cys gatgaa aacgetttg tgctttaacact gttggagga cacaactgt gtc 1536 AspGlu AsnAlaLeu CysPheAsnThr ValGlyGly HisAsnCys Val tgcaag cctggctac accgggaatgga accacgtgc aaagetttc tgc 1584 CysLys ProGlyTyr ThrGlyAsnGly ThrThrCys LysAlaPhe Cys aaagat ggctgtaga aacggaggagcg tgcattget gccaatgtg tgt 1632 LysAsp GlyCysArg AsnGlyGlyAla CysIleAla AlaAsnVal Cys gcctgc ccacaaggc ttcacgggaccc agctgtgag acagacatt gac 1680 AlaCys ProGlnGly PheThrGlyPro SerCysGlu ThrAspIle Asp gagtgc tctgagggc tttgttcagtgt gacagccgt gecaactgc atc 1728 GluCysSer Glu GlnCys Asp Ala Cys Ile Gly Ser Asn Phe Arg Val aacctg gggtggtat cactgtgag tgcaga ggctaccat gac 1776 cct gac AsnLeu GlyTrpTyr HisCysGlu CysArg Gly His Asp Pro Asp Tyr aatgggatg tttgcgcca ggcggagaa tcctgtgaa gatattgac gaa 1824 AsnGlyMet PheAlaPro GlyGlyGlu SerCysGlu AspIleAsp Glu tgcgggact gggaggcac agctgcacc aacgacacc atttgcttc aac 1872 CysGlyThr GlyArgHis SerCysThr AsnAspThr IleCysPhe Asn ttggacggg ggatacgat tgccggtgt ccccatggg aagaactgc act 1920 LeuAspGly GlyTyrAsp CysArgCys ProHisGly LysAsnCys Thr ggggactgc gtgcacgag gggaaagtg aagcacacc ggccagatc tgg 1968 GlyAspCys ValHisGlu GlyLysVal LysHisThr GlyGlnIle Trp gtgctggaa aacgacagg tgctccgtg tgttcctgg cagactggg ttt 2016 ValLeuGlu AsnAspArg CysSerVal CysSerTrp GlnThrGly Phe gtcatgtgt cgacggatg gtctgcgac tgcgaaaac cccacagat gac 2064 ValMetCys ArgArgMet ValCysAsp CysGluAsn ProThrAsp Asp ctttcctgc tgccctgag tgtgaccca aggctgagc agtcagtgc ctg 2112 LeuSerCys CysProGlu CysAspPro ArgLeuSer SerGlnCys Leu catcaaaac ggggaaacc gtgtacaac agcggcgac acctgggtc cag 2160 HisGlnAsn GlyGluThr ValTyrAsn SerGlyAsp ThrTrpVal Gln gattgccgt cagtgccgc tgcttgcaa ggagaagtt gactgttgg ccc 2208 AspCysArg GlnCysArg CysLeuGln GlyGluVal AspCysTrp Pro ctggettgc ccagaggta gaatgtgaa tttagcgtc cttcctgag aac 2256 LeuAlaCys ProGluVal GluCysGlu PheSerVal LeuProGlu Asn gagtgctgc ccacgctgt gtcacc ccttgtcag gccgacacc atc 2304 gat GluCysCys ProArgCys ValThr ProCysGln AspThr Ile Asp Ala cgc gac accaaa acctgc gagatg aacgtg cgc 2352 aat atc ctg gtt gac Arg ThrLys ThrCys GluMet Val Asn Leu Asn Val Asp Asp Arg Ile ttc acc ggg tct tcc tgg atc aag cac ggc acg gag tgt acc ctc tgc 2400 Phe Thr Gly Ser Ser Trp Ile Lys His Gly Thr Glu Cys Thr Leu Cys cag tgc aag aat ggc cat ttg tgc tgc tca gtg gat cca cag tgc ctt 2448 Gln Cys Lys Asn Gly His Leu Cys Cys Ser Val Asp Pro Gln Cys Leu cag gag ctg tga 2460 Gln Glu Leu <210> 12 <211> 819 <212> PRT
<213> Mus musculus <400> 12 Met His Ala Met Glu Ser Arg Val Leu Leu Arg Thr Phe Cys Val Ile , 15 Leu Gly Leu Gly Ala Val Trp Gly Leu Gly Val Asp Pro Ser Leu Gln Ile Asp Val Leu Thr Glu Leu Glu Leu Gly Glu Ser Thr Asp Gly Val Arg Gln Val Pro Gly Leu His Asn Gly Thr Lys Ala Phe Leu Phe Gln Glu Ser Pro Arg Ser Ile Lys Ala Ser Thr Ala Thr Ala Glu Arg Phe Leu Gln Lys Leu Arg Asn Lys His Glu Phe Thr Ile Leu Val Thr Leu Lys Gln Ile His Leu Asn Ser Gly Val Ile Leu Ser Ile His His Leu Asp His Arg Tyr Leu Glu Leu Glu Ser Ser Gly His Arg Asn Glu Ile Arg Leu His Tyr Arg Ser Gly Thr His Arg Pro His Thr Glu Val Phe 130 l35 140 Pro Tyr Ile Leu Ala Asp Ala Lys Trp His Lys Leu Ser Leu Ala Phe 145 150 155 160 ~' Ser Ala Ser His Leu Ile Leu His Ile Asp Cys Asn Lys Ile Tyr Glu Arg Val Val Glu Met Pro Phe Thr Asp Leu Ala Leu Gly Thr Thr Phe Trp Leu Gly Gln Arg Asn Asn Ala His Gly Tyr Phe Lys Gly Ile Met Gln Asp Val His Val Leu Val Met Pro Gln Gly Phe Ile Ala Gln Cys 210 2l5 220 Pro Asp Leu Asn Arg Thr Cys Pro Thr Cys Asn Asp Phe His Gly Leu Val Gln Lys Ile Met Glu Leu Gln Asp Ile Leu Ser Lys Thr Ser Ala Lys Leu Ser Arg Ala Glu Gln Arg Met Asn Arg Leu Asp Gln Cys Tyr Cys Glu Arg Thr Cys Thr Val Lys Gly Thr Thr Tyr Arg Glu Ser Glu Ser Trp Thr Asp Gly Cys Lys Asn Cys Thr Cys Leu Asn Gly Thr Ile Gln Cys Glu Thr Leu Val Cys Pro Ala Pro Asp Cys Pro Pro Lys Ser Ala Pro Ala Tyr Val Asp Gly Lys Cys Cys Lys Glu Cys Lys Ser Thr Cys Gln Phe Gln Gly Arg Ser Tyr Phe Glu Gly Glu Arg Asn Thr Ala Tyr Ser Ser Ser Gly Met Cys Val Leu Tyr Glu Cys Lys Asp Gln Thr Met Lys Leu Val Glu Asn Ile Gly Cys Pro Pro Leu Asp Cys Pro Glu Ser His Gln Ile Ala Leu Ser His Ser Cys Cys Lys Val Cys Lys Gly Tyr Asp Phe Cys Ser Glu Lys His Thr Cys Met Glu Asn Ser V'al Cys Arg Asn Leu Asn Asp Arg Val Val Cys Ser Cys Arg Asp Gly Phe Arg Ala Leu Arg Glu Asp Asn Ala Tyr Cys Glu Asp Ile Asp Glu Cys Ala Glu Gly Arg His Tyr Cys Arg Glu Asn Thr Met Cys Val Asn Thr Pro Gly Ser Phe Met Cys Val Cys Lys Thr Gly Tyr Ile Arg Ile Asp Asp Tyr Ser Cys Thr Glu His Asp Glu Cys Leu Thr Thr Gln His Asn Cys Asp Glu Asn Ala Leu Cys Phe Asn Thr Val Gly Gly His Asn Cys Val Cys Lys Pro Gly Tyr Thr Gly Asn Gly Thr Thr Cys Lys Ala Phe Cys Lys Asp Gly Cys Arg Asn Gly Gly Ala Cys Ile Ala Ala Asn Val Cys Ala Cys Pro Gln Gly Phe Thr Gly Pro Ser Cys Glu Thr Asp Ile Asp Glu Cys Ser Glu Gly Phe Val Gln Cys Asp Ser Arg Ala Asn Cys Ile Asn Leu Pro Gly Trp Tyr His Cys Glu Cys Arg Asp Gly Tyr His Asp Asn Gly Met Phe Ala Pro Gly Gly Glu Ser Cys Glu Asp Ile Asp Glu Cys Gly Thr Gly Arg His Ser Cys Thr Asn Asp Thr Ile Cys Phe Asn Leu Asp Gly Gly Tyr Asp Cys Arg Cys Pro His Gly Lys Asn Cys Thr Gly Asp Cys Val His Glu Gly Lys Val Lys His Thr Gly Gln Ile Trp Val Leu Glu Asn Asp Arg Cys Ser Val Cys Ser Trp Gln Thr Gly Phe Val Met Cys Arg Arg Met Val Cys Asp Cys Glu Asn Pro Thr Asp Asp Leu Ser Cys Cys Pro Glu Cys Asp Pro Arg Leu Ser Ser Gln Cys Leu His Gln Asn Gly Glu Thr Val Tyr Asn Ser Gly Asp Thr Trp Val Gln Asp Cys Arg Gln Cys Arg Cys Leu Gln Gly Glu Val Asp Cys Trp Pro Leu Ala Cys Pro Glu Val Glu Cys Glu Phe Ser Val Leu Pro Glu Asn Glu Cys Cys Pro Arg Cys Val Thr Asp Pro Cys Gln Ala Asp Thr Ile Arg Asn Asp Ile Thr Lys Thr Cys Leu Asp Glu Met Asn Val Val Arg Phe Thr Gly Ser Ser Trp Ile Lys His Gly Thr Glu Cys Thr Leu Cys Gln Cys Lys Asn Gly His Leu Cys Cys Ser Val Asp Pro Gln Cys Leu 805 8l0 815 Gln Glu Leu <210>

<211>

<212>
DNA

<213> s Gallu gallus <220>

<221>
CDS

<222> .(2453) (1)..

<400>

atg gag ggc tgcggctta ggcacgctt tgccttCtC CtCtgcctg 48 tcc Met Glu Gly CysGlyLeu GlyThrLeu CysLeuLeu LeuCysLeu Ser ggg cca gta ggcttcggc gtggacccc tcgctgcag atcgacgtg 96 gtc Gly Pro Val GlyPheGly ValAspPro SerLeuGln IleAspVal Val ctg tec ctg gggctgccg ggctacgcg gcgggcgtg cgccaggtg 144 gag Leu Ser Leu GlyLeuPro GlyTyrAla AlaGlyVal ArgGlnVal Glu ccg ggg cac aacgggagc aaagccttc ctcttccca gatacttca 192 ctg Pro Gly His AsnGlySer LysAlaPhe LeuPhePro AspThrSer Leu aga agt aag gcgtctcca gaaacaget gaaatcttt tttcagaag 240 gta Arg Ser Lys AlaSerPro GluThrAla GluIlePhe PheGlnLys Val ttg aga aaa tatgaattc acaatcctg gtgacctta aaacaagcc 288 aat Leu Arg Lys TyrGluPhe ThrIleLeu ValThrLeu LysGlnAla Asn catttaaat tcaggggtt attttctct attcatcac gatcac agg 336 tta HisLeuAsn GlyVal IlePheSer IleHisHisLeu AspHis Arg Ser tatctggaa ttggaaagc agcggtcat cgaaatgaaatc aggttg cat 384 TyrLeuGlu LeuGluSer SerGlyHis ArgAsnGluIle ArgLeu His taccgtaca ggcagtcat cgctcccac acagaagtattc ccatac atc 432 TyrArgThr GlySerHis ArgSerHis ThrGluValPhe ProTyr Ile ctggcagac gataagtgg cacaggctt tccttagcaatc agtgcc tct 480 LeuAlaAsp AspLysTrp HisArgLeu SerLeuAlaIle SerAla Ser cacttgatt ttacacgtg gactgcaat aaaatctatgaa agagtt gtg 528 HisLeuIle LeuHisVal AspCysAsn LysIleTyrGlu ArgVal Val gagaagccc ttcatggac ttacctgtg ggtacaaccttt tggcta gga 576 GluLysPro PheMetAsp LeuProVal GlyThrThrPhe TrpLeu Gly cagaggaat aatgcacac ggttatttt aagggcataatg caagat gtg 624 GlnArgAsn AsnAlaHis GlyTyrPhe LysGlyIleMet GlnAsp Val caattactt gtcatgcct caaggattt atttctcagtgc ccagat ctt 672 GlnLeuLeu ValMetPro GlnGlyPhe IleSerGlnCys ProAsp Leu aatcggaca tgcccaact tgtaatgat ttccatggactt gtgcag aaa 720 AsnArgThr CysProThr CysAsnAsp PheHisGlyLeu ValGln Lys attatggaa ctgcaagac attttaget aaaacgtcaget aagctg tcg 768 IleMetGlu LeuGlnAsp IleLeuAla LysThrSerAla LysLeu Ser caagetgag cagaggatg aacaagttg gatcagtgctat tgtgaa agg 816 GlnAlaGlu GlnArgMet AsnLysLeu AspGlnCysTyr CysGlu Arg acctgcaca atgaaaggc atgacatac agagaatttgaa tcctgg aca 864 ThrCysThr MetLysGly MetThrTyr GluPheGlu SerTrp Thr Arg gatggttgt aag tgc acttgc ggcactgtg cagtgt gaa 912 aac atg aat Asp Cys Lys Cys ThrCys ThrVal Gln Glu Gly Asn Met Cys Asn Gly getttgatt tgctcactc tctgaa caacataat tctgccctg tca 960 tgt AlaLeuIle CysSerLeu Ser ProProAsn SerAlaLeu Ser Asp Cys tacgtggat ggaaagtgc tgcaaagaa tgtaaatcg gtgtgcata ttt 1008 TyrValAsp Gly Cys CysLysGlu CysGlnSer ValCysIle Phe Lys gaaggaaga acctaattt gaaggacaa agagaaacg gtgtattca aga 1056 GluGlyArg ThrTyrPhe GluGlyGln ArgGluThr ValTyrSer Ser taaggggac tgtgttctg tttgagtga aaggacaaa aaaatgcag agt 1104 SerGlyAsp CysValLeu PheGluCys LysAspHis LysMetGln Arg attecaaaa gacagttgt gaaactttg aactgcccg gaatctcaa cag 1152 IleProLys AspSerCys AlaThrLeu AsnCysPro GluSerGln Gln atcacatta tctcacagt tgatgaaaa atctgtaaa ggccatgac ttt 1200 IleProLeu SerHisSer CysCysLys IleCysLys GlyHisAsp Phe tgcactgaa ggacataac tgtatggag cattatgta tgccgaaac cta 1248 CysThrGlu GlyHisAsn CysMetGlu HisSerVal CysArgAsn Leu gatgacaga getgtatgt agatgaaga gatggcttc cgggccctt cgg 1296 AspAspArg AlaValCys SerCysArg AspGlyPhe ArgAlaLeu Arg gaggacaat gcatactgt gaagatgtt gatgagtgt gccgagggg cag 1344 GluAspAsn AlaTyrCys GluAspVal AspGluCys AlaGluGly Gln cactactgt cgggagaac accatgtgt gtaaataaa caaggatac tta 1392 HisTyrCys ArgGluAsn ThrMetCys ValAsnThr ProGlySer Phe atgtgcata tgaaaaaca ggatatata agaattgat gactattaa tgt 1440 MetCysIle CysLysThr GlyTyrIle ArgIleAsp AspTyrSer Cys acagagcac gatgaatgt gtaaaaaac cagcaaaac tgtgatgaa aat 1488 ThrGluHis AspGluCys ValThrAsn GlnHisAsn CysAspGlu Asn gcgatatgt ttcaacacg gtgggtggg cacaactgt gtctgcaag atg 1536 AlaLeuCys PheAsnThr ValGlyGly AsnCys Cys Leu His Val Lys ggttacaca ggaaat acggtgtgt gcattt tgaaaa 1584 ggg aaa gat ggg GlyTyrThr AsnGly Cys LysAlaPhe CysLysAsp Gly Gly Thr Val tgcaggaat ggaggagcc tgtattget tccaacgtg tgtgcctgc cca 1632 CysArgAsn GlyGlyAla CysIleAla SerAsnVal CysAlaCys Pro caaggcttc actggcccc agctgtgaa actgacatt gatgaatgc tct 1680 GlnGlyPhe Thr.GlyPro SerCysGlu ThrAspIle AspGluCys Ser gatggcttt gtgcagtgt gacagccgt getaattgc atcaatctg cca 1728 AspGlyPhe ValGlnCys AspSerArg AlaAsnCys IleAsnLeu Pro gggtggtac cactgtgaa tgcagggat ggctaccat gacaatggg atg 1776 GlyTrpTyr HisCysGlu CysArgAsp GlyTyrHis AspAsnGly Met ttttcacca agtggagaa tcctgtgaa gacattgat gaatgtgca act 1824 PheSerPro SerGlyGlu SerCysGlu AspIleAsp GluCysAla Thr ggaaggcat agctgtgcc aatgacact gtttgcttt aacctggat ggt 1872 GlyArgHis SerCysAla AsnAspThr ValCysPhe AsnLeuAsp Gly gggtatgac tgtcgatgt ccacatggc aagaactgc acaggagac tgt 1920 GlyTyrAsp CysArgCys ProHisGly LysAsnCys ThrGlyAsp Cys atccatgaa gacaaaatc aagcacaat ggtcagatt tgggtgctg gag 1968 IleHisGlu AspLysIle LysHisAsn GlyGlnIle TrpValLeu Glu aacgacaga tgctctgtc tgctcatgc cagagtgga tacgtgatg tgc 2016 AsnAspArg CysSerVal CysSerCys GlnSerGly TyrValMet Cys cggcgaatg gtctgtgac tgtgaaaat cccactgtt gacctcttt tgc 2064 ArgArgMet ValCysAsp CysGluAsn ProThrVal AspLeuPhe Cys tgtcctgag tgtgaccca aggctcagc agtcaatgt ttacatcag agt 2112 CysProGlu CysAspPro ArgLeuSer SerGlnCys LeuHisGln Ser ggggagctt tcctacaac agtggtgac tcctggata caaaactgt cag 2160 GlyGluLeu SerTyrAsn SerGlyAsp SerTrpIle GlnAsnCys Gln cagtgtcgc tgcttgcaa ggagag gactgttgg cccttaccg tgc 2208 gtt GlnCysArg CysLeuGln GlyGlu CysTrp ProLeuPro Cys Val Asp ccagaggtagac tgtgagttc agtgtcctc cctgagaat gagtgctgc 2256 ProGluValAsp CysGluPhe SerValLeu ProGluAsn GluCysCys ccacgctgtgtc actgacccc tgccaagcg gacaccate cgtaatgac 2304 ProArgCysVal ThrAspPro CysGlnAla AspThrIle ArgAsnAsp atcaccaaaacc tgcctggat gaaaccaat gttgttcgc ttcactgga 2352 IleThrLysThr CysLeuAsp GluThrAsn ValValArg PheThrGly tcttcttggatt aagcatggc acagagtgc acactctgc caatgtaag 2400 SerSerTrpIle LysHisGly ThrGluCys ThrLeuCys GlnCysLys aatggccacgtc tgttgctca gtggatcca cagtgcctt caggaactg 2448 AsnGlyHisVal CysCysSer ValAspPro GlnCysLeu GlnGluLeu tgaca <210> 14 <211> 816 <212> PRT
<213> Callus gallus <400> 14 Met Glu Ser Gly Cys Gly Leu Gly Thr Leu Cys Leu Leu Leu Cys Leu Gly Pro Val Val Gly Phe Gly Val Asp Pro Ser Leu Gln Ile Asp Val Leu Ser Glu Leu Gly Leu Pro Gly Tyr Ala Ala Gly Val Arg Gln Val Pro Gly Leu His Asn Gly Ser Lys Ala Phe Leu Phe Pro Asp Thr Ser Arg Ser Val Lys Ala Ser Pro Glu Thr Ala Glu Ile Phe Phe Gln Lys Leu Arg Asn Lys Tyr Glu Phe Thr Ile Leu Val Thr Leu Lys Gln Ala His Leu Asn Ser Gly Val Ile Phe Ser Ile His His Leu Asp His Arg Tyr Leu Glu Leu Glu Ser Ser Gly His Arg Asn Glu Ile Arg Leu His Tyr Arg Thr Gly Ser His Arg Ser His Thr Glu Val Phe Pro Tyr Ile Leu Ala Asp Asp Lys Trp His Arg Leu Ser Leu Ala Ile Ser Ala Ser l60 His Leu Ile Leu His Val Asp Cys Asn Lys Ile Tyr Glu Arg Val Val Glu Lys Pro Phe Met Asp Leu Pro Val Gly Thr Thr Phe Trp Leu Gly Gln Arg Asn Asn Ala His Gly Tyr Phe Lys Gly Ile Met Gln Asp Val Gln Leu Leu Val Met Pro Gln Gly Phe Ile Ser Gln Cys Pro Asp Leu Asn Arg Thr Cys Pro Thr Cys Asn Asp Phe His Gly Leu Val Gln Lys Ile Met Glu Leu Gln Asp Ile Leu Ala Lys Thr Ser Ala Lys Leu Ser Gln Ala Glu Gln Arg Met Asn Lys Leu Asp Gln Cys Tyr Cys Glu Arg Thr Cys Thr Met Lys Gly Met Thr Tyr Arg Glu Phe Glu Ser Trp Thr Asp Gly Cys Lys Asn Cys Thr Cys Met Asn Gly Thr Val Gln Cys Glu Ala Leu Ile Cys Ser Leu Ser Asp Cys Pro Pro Asn Ser Ala Leu Ser 305 310 3l5 320 Tyr Val Asp Gly Lys Cys Cys Lys Glu Cys Gln Ser Val Cys Ile Phe Glu Gly Arg Thr Tyr Phe Glu Gly Gln Arg Glu Thr Val Tyr Ser Ser Ser Gly Asp Cys Val Leu Phe Glu Cys Lys Asp His Lys Met Gln Arg Ile Pro Lys Asp Ser Cys Ala Thr Leu Asn Cys Pro Glu Ser Gln Gln Ile Pro Leu Ser His Ser Cys Cys Lys Ile Cys Lys Gly His Asp Phe Cys Thr Glu Gly His Asn Cys Met Glu His Ser Val Cys Arg Asn Leu Asp Asp Arg Ala Val Cys Ser Cys Arg Asp Gly Phe Arg Ala Leu Arg Glu Asp Asn Ala Tyr Cys Glu Asp Val Asp Glu Cys Ala Glu Gly Gln His Tyr Cys Arg Glu Asn Thr Met Cys Val Asn Thr Pro Gly Ser Phe Met Cys Ile Cys Lys Thr Gly Tyr Ile Arg Ile Asp Asp Tyr Ser Cys Thr Glu His Asp Glu Cys Val Thr Asn Gln His Asn Cys Asp Glu Asn Ala Leu Cys Phe Asn Thr Val Gly Gly His Asn Cys Val Cys Lys Leu Gly Tyr Thr Gly Asn Gly Thr Val Cys Lys Ala Phe Cys Lys Asp Gly Cys Arg Asn Gly Gly Ala Cys Ile Ala Ser Asn Val Cys Ala Cys Pro Gln Gly Phe Thr Gly Pro Ser Cys Glu Thr Asp Ile Asp Glu Cys Ser Asp Gly Phe Val Gln Cys Asp Ser Arg Ala Asn Cys Ile Asn Leu Pro Gly Trp Tyr His Cys Glu Cys Arg Asp Gly Tyr His Asp Asn Gly Met Phe Ser Pro Ser Gly Glu Ser Cys Glu Asp Ile Asp Glu Cys Ala Thr Gly Arg His Ser Cys Ala Asn Asp Thr Val Cys Phe Asn Leu Asp Gly Gly Tyr Asp Cys Arg Cys Pro His Gly Lys Asn Cys Thr Gly Asp Cys Ile His Glu Asp Lys Ile Lys His Asn Gly Gln Ile Trp Val Leu Glu Asn Asp Arg Cys Ser Val Cys Ser Cys Gln Ser Gly Tyr Val Met Cys Arg Arg Met Val Cys Asp Cys Glu Asn Pro Thr Val Asp Leu Phe Cys Cys Pro Glu Cys Asp Pro Arg Leu Ser Ser Gln Cys Leu His Gln Ser Gly Glu Leu Ser Tyr Asn Ser Gly Asp Ser Trp Ile Gln Asn Cys Gln Gln Cys Arg Cys Leu Gln Gly Glu Val Asp Cys Trp Pro Leu Pro Cys Pro Glu Val Asp Cys Glu Phe Ser Val Leu Pro Glu Asn Glu Cys Cys Pro Arg Cys Val Thr Asp Pro Cys Gln Ala Asp Thr Ile Arg Asn Asp Ile Thr Lys Thr Cys Leu Asp Glu Thr Asn Val Val Arg Phe Thr Gly Ser Ser Trp Ile Lys His Gly Thr Glu Cys Thr Leu Cys Gln Cys Lys Asn Gly His Val Cys Cys Ser Val Asp Pro Gln Cys Leu Gln Glu Leu

Claims (155)

WHAT IS CLAIMED IS:

1. ~A method of expressing a functional peptide in an insect cell, said method comprising:
providing a nucleic acid construct including at least a nucleic acid encoding at least a NELL1 peptide in frame with a nucleic acid encoding an insect secretory signal peptide;
transfecting an insect cell with said nucleic acid construct;
culturing said insect cell under conditions that permit expression of the NELL1 peptide;
optionally collecting NELL1 peptide secreted from the cell line;
optionally substantially purifying the NELL1 peptide; and optionally testing the activity of the NELL1 peptide.

2. ~The method of claim 1, wherein said insect cell is a high five cell.

3. ~The method of claim 1, wherein said insect secretory signal peptide is a melittin signal sequence.

4. ~The method of claim 1, wherein the nucleic acid encoding NELL1 is selected from the group comprising: SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.

5. ~The method of claim 1, wherein the NELL1 peptide is selected from the group comprising: SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

6. ~A nucleic acid construct for expressing a NELL1 peptide in an insect cell, said nucleic acid construct comprising at least a nucleic acid encoding at least a NELL1 peptide in frame with a nucleic acid encoding an insect secretory signal peptide.

7. ~The nucleic acid of claim 5, wherein said insect secretory signal peptide is a melittin signal sequence.

8. ~The nucleic acid of claim 5, wherein the nucleic acid encoding NELL1 is selected from the group comprising: SEQ ID NO:1, SEQ ID NO:3, or SEQ ID
NO:5.

9. ~The nucleic acid of claim 5, wherein the NELL1 peptide is selected from the group comprising: SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO :6.

10. ~A cell line for expressing a functional NELL1 peptide, said cell line including a nucleic acid construct comprising at least a nucleic acid encoding at least a NELL1 peptide in frame with a nucleic acid encoding an insect secretory signal peptide.

11. ~The cell of claim 10, wherein said cell is an insect cell.

12. ~The cell of claim 11, wherein said cell is a high five cell.

13. ~The cell of claim 10, wherein said cell secretes said NELL1 peptide.

14. ~The cell of claim 10, wherein said secretory signal peptide is a melittin signal sequence.

15. ~The cell of claim 10, wherein the nucleic acid encoding NELL1 peptide is selected from the group comprising: SEQ ID NO:1, SEQ ID NO:3, or SEQ ID
NO:5.

16. ~The method of claim 1, wherein the NELL1 peptide is selected from the group comprising: SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO :6.

17. ~A polypeptide comprising a NELL1 peptide and an insect secretory signal peptide.

18. ~The polypeptide of claim 16, wherein the NELL1 peptide is selected from the group comprising: SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

19. ~The polypeptide of claim 17, wherein said secretory signal peptide is a melittin signal sequence.

20. ~A method of expressing a functional peptide in a mammalian cell, said method comprising:
providing a nucleic acid construct including at least a nucleic acid encoding at least a NELL1 peptide in frame with a nucleic acid encoding a secretory signal peptide;~
transfecting a mammalian cell with said nucleic acid construct;
culturing said mammalian cell under conditions that permit expression of the NELL1 peptide;
optionally collecting NELL1 peptide secreted from the cell line;
optionally substantially purifying the NELL1 peptide; and optionally testing the activity of the NELL1 peptide to induce bone formation.

21. The method of claim 20, wherein said insect cell is a COS7 cell.

22. The method of claim 20, wherein said insect secretory signal peptide is a NELL peptide signal sequence.

23. The method of claim 20, wherein the nucleic acid encoding NELL1 is selected from the group comprising: SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.

24. The method of claim 20, wherein the NELL1 peptide is selected from the group comprising: SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

25. A nucleic acid construct for expressing a NELL1 peptide in a mammalian cell, said nucleic acid construct comprising at least a nucleic acid encoding at least a NELL1 peptide in frame with a nucleic acid encoding a secretory signal peptide.

26. The nucleic acid of claim 24, wherein said insect secretory signal peptide is a NELL peptide signal sequence.

27. The nucleic acid of claim 24, wherein the nucleic acid encoding NELL1 is selected from the group comprising: SEQ ID NO:1, SEQ ID NO:3, or SEQ ID
NO:5.

28. The nucleic acid of claim 24, wherein the NELL1 peptide is selected from the group comprising: SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO :6.

29. A cell line for expressing a functional NELL1 peptide, said cell line including a nucleic acid construct comprising at least a nucleic acid encoding at least a NELL1 peptide in frame with a nucleic acid encoding a secretory signal peptide.

30. The cell of claim 29, wherein said cell is a mammalian cell.

31. The cell of claim 30, wherein said cell is a COS7 cell.

32. The cell of claim 29, wherein said cell secretes said NELL1 peptide.

33. The cell of claim 29, wherein said secretory signal peptide is a NELL
peptide signal sequence.

34. The cell of claim 29, wherein the nucleic acid encoding NELL1 peptide is selected from the group comprising: SEQ ID NO:1, SEQ ID NO:3, or SEQ ID
NO:5.

35. The method of claim 20, wherein the NELL1 peptide is selected from the group comprising: SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO :6.

36. A polypeptide comprising a NELL1 peptide and a secretory signal peptide.

37. The polypeptide of claim 35, wherein the NELL1 peptide is selected from the group comprising: SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

38. The polypeptide of claim 35, wherein said secretory signal peptide is a NELL peptide signal sequence.

39. A method of expressing a functional peptide in an insect cell, said method comprising:
providing a nucleic acid construct including at least a nucleic acid encoding at least a NELL2 peptide and an insect secretory signal peptide in frame with the NELL2 peptide;
transfecting an insect cell with said nucleic acid construct ;
culturing said insect cell under conditions that permit expression of the NELL2 peptide;
optionally collecting NELL2 peptide secreted from the cell line;
optionally substantially purifying the NELL2 peptide; and optionally testing the activity of the NELL2 peptide.

40. The method of claim 39, wherein said insect cell is a high five cell.

41. The method of claim 39, wherein said insect secretory signal peptide is a melittin signal sequence.

42. The method of claim 39, wherein the nucleic acid encoding NELL2 is selected from the group comprising: SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:13.

43. The method of claim 39, wherein the NELL2 peptide is selected from the group comprising: SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:
14.

44. A nucleic acid construct for expressing a NELL2 peptide in an insect cell, said nucleic acid construct comprising at least a nucleic acid encoding at least a NELL2 peptide in frame with a nucleic acid encoding an insect secretory signal peptide.

45. The nucleic acid of claim 43, wherein said insect secretory signal peptide is a melittin signal sequence.

46. The nucleic acid of claim 43, wherein the nucleic acid encoding NELL2 is selected from the group comprising: SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:13.

47. The nucleic acid of claim 5, wherein the NELL2 peptide is selected from the group comprising: SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ
ID NO:14.

48. A cell line for expressing a functional NELL2 peptide, said cell line including a nucleic acid construct comprising at least a nucleic acid encoding at least a NELL2 peptide in frame with a nucleic acid encoding an insect secretory signal peptide.

49. The cell of claim 48, wherein said cell is an insect cell.

50. The cell of claim 49, wherein said cell is a high five cell.

51. The cell of claim 48, wherein said cell secretes said NELL2 peptide.

52. The cell of claim 48, wherein said secretory signal peptide is a melittin signal sequence.

53. The cell of claim 48, wherein the nucleic acid encoding NELL2 peptide is selected from the group comprising: SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:11, or SEQ ID NO:13.

54. The method of claim 39, wherein the NELL2 peptide is selected from the group comprising: SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID
NO:14.

55. A polypeptide comprising a NELL2 peptide and an insect secretory signal peptide.

56. The polypeptide of claim 55, wherein the NELL2 peptide is selected from the group comprising: SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ
ID NO:14.

57. The polypeptide of claim 55, wherein said secretory signal peptide is a melittin signal sequence.

58. A method of expressing a functional peptide in a mammalian cell, said method comprising:
providing a nucleic acid construct including at least a nucleic acid encoding at least a NELL2 peptide and a nucleic acid encoding a secretory signal peptide in frame with the NELL2 peptide;
transfecting an insect cell with said nucleic acid construct;
culturing said insect cell under conditions that permit expression of the NELL2 peptide;
optionally collecting NELL2 peptide secreted from the cell line;
optionally substantially purifying the NELL2 peptide; and optionally testing the activity of the NELL2 peptide to promote neuronal cell survival.

59. The method of claim 58, wherein said mammalian cell is a COS7 cell.

60. The method of claim 58, wherein said mammalian secretory signal peptide is a NELL peptide signal sequence.

61. The method of claim 58, wherein the nucleic acid encoding NELL1 is selected from the group comprising: SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:13.

62. The method of claim 58, wherein the NELL2 peptide is selected from the group comprising: SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID
NO:14.

63. A nucleic acid construct for expressing a NELL2 peptide in a mammalian cell, said nucleic acid construct comprising at least a nucleic acid encoding at least a NELL2 peptide in frame with a nucleic acid encoding a secretory signal peptide.

64. The nucleic acid of claim 63, wherein said mammalian secretory signal peptide is a NELL peptide signal sequence.

65. The nucleic acid of claim 63, wherein the nucleic acid encoding NELL2 is selected from the group comprising: SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:11, or SEQ ID NO:13.

66. The nucleic acid of claim 63, wherein the NELL2 peptide is selected from the group comprising: SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ
ID NO:14.

67. A cell line for expressing a functional NELL2 peptide, said cell line including a nucleic acid construct comprising at least a nucleic acid encoding at least a NELL2 peptide in frame with a nucleic acid encoding a secretory signal peptide.

68. The cell of claim 67, wherein said cell is a mammalian cell.

69. The cell of claim 68, wherein said cell is a COS7 cell.

70. The cell of claim 67, wherein said cell secretes said NELL2 peptide.

71. The cell of claim 67, wherein said secretory signal peptide is a NELL
peptide signal sequence.

72. The cell of claim 67, wherein the nucleic acid encoding NELL2 peptide is selected from the group comprising: SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:11, or SEQ ID NO:13.

73. The method of claim 58, wherein the NELL2 peptide is selected from the group comprising: SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID
NO:14.

74. A method of increasing osteogenic cell differentiation comprising:
increasing the concentration of a NELL1 gene product in an osteogenic cell;
optionally applying a second osteogenic agent; and inducing the expression of cellular marker of osteoblastic differentiation.

75. The method of claim 74, wherein the increasing the concentration of a NELL1 gene product comprises applying a NELL1 peptide to an osteogenic cell.

76. The method of claim 74 wherein the NELL1 peptide is selected from the group comprising: SEQ ID NO:2, SEQ ID NO: 4, or SEQ ID NO:6.

77. The method of claim 74, wherein the increasing the concentration of a NELL1 gene product comprises inducing the expression of an endogenous NELL1 gene.

78. The method of claim 77 wherein increasing the concentration of a NELL1 gene product comprises increasing cellular levels of Cbfa1.

79. The method of claim 74, wherein the increasing the concentration of a NELL1 gene product comprises transfecting the osteogenic cell with a nucleic acid construct encoding a NELL1 peptide.

80. The method of claim 74 wherein the nucleic acid construct encoding a NELL1 peptide comprises at least one selected from the group: SEQ ID NO:1, SEQ
ID NO: 3, or SEQ ID NO:5.

81. The method of claim 74 wherein the NELL1 peptide is selected from the group comprising: SEQ ID NO:2, SEQ ID NO: 4, or SEQ ID NO:6.

82. The method of claim 74 wherein the osteogenic cell is selected from the group comprising: an osteoblast, a mesenchymal cell, a fibroblast, fetal embryonic cell, a stem cell, a bone marrow cell, a dura cell, a chondrocytes, a chondroblast, primary calvarial cells, stem cells, adipose stem cells.

83. The method of claim 74, wherein the second osteogenic peptide is selected from the group comprising: TGF-.beta., BMP2, BMP4, BMP7, bFGF, collagen.

84. The method of claim 74 wherein the induced cellular marker of osteoblastic differentiation is selected from the group comprising: alkaline phosphatase activity, osteocalcin mRNA expression, osteoponin mRNA expression, decorin expression, and laminin B expression.

85. A method of increasing osteoblastic mineralization comprising:
increasing the concentration of a NELL1 gene product in an osteogenic cell;

optionally applying an agent to the osteogenic cell; and inducing the expression of cellular marker of mineralization.

86. The method of claim 85, wherein the increasing the concentration of a NELL1 gene product comprises applying a NELL1 peptide to an osteogenic cell.

87. The method of claim 85 wherein the NELL1 peptide is selected from the group comprising: SEQ ID NO:2, SEQ ID NO: 4, or SEQ ID NO:6.

88. The method of claim 85, wherein the increasing the concentration of a NELL1 gene product comprises inducing the expression of an endogenous NELL1 gene.

89. The method of claim 88 wherein increasing the concentration of a NELL1 gene product comprises increasing cellular levels of Cbfa1.

90. The method of claim 85, wherein the increasing the concentration of a NELL1 gene product comprises transfecting the osteogenic cell with a nucleic acid construct encoding a NELL1 peptide.

91. The method of claim 85 wherein the nucleic acid construct encoding a NELL1 peptide comprises at least one selected from the group: SEQ ID NO:1, SEQ
ID NO: 3, or SEQ ID NO:5.

92. The method of claim 85 wherein the NELL1 peptide is selected from the group comprising: SEQ ID NO:2, SEQ ID NO: 4, or SEQ ID NO:6.

93. The method of claim 85 wherein the osteogenic cell is selected from the group comprising: an osteoblast, a mesenchymal cell, a fibroblast, fetal embryonic cell, a stem cell, a bone marrow cell, a dura cell, a chondrocytes, a chondroblast, primary calvarial cells, stem cells, adipose stem cells.

94. The method of claim 85, wherein the second osteogenic agent is selected from the group comprising: TGF-.beta., BMP2, BMP4, BMP7, bFGF, collagen.

95. The method of claim 85 wherein the induced cellular marker of mineralization is selected from the group comprising: calcium incorporation.

96. A method of increasing intramembraneous bone formation comprising:
increasing the concentration of a NELL1 gene product in a region where bone formation is desired;

optionally applying a second agent to approximately the same region where bone formation is desired; and inducing the formation of intramembraneous bone formation.

97. The method of claim 96, wherein the increasing the concentration of a NELL1 gene product comprises applying a NELL1 peptide to the region where bone formation is desired.

98. The method of claim 96 wherein the NELL1 peptide is selected from the group comprising: SEQ ID NO:2, SEQ ID NO: 4, or SEQ ID NO:6.

99. The method of claim 96, wherein the increasing the concentration of a NELL1 gene product comprises inducing the expression of an endogenous NELL1 gene.

100. The method of claim 96, wherein the increasing the concentration of a NELL1 gene product comprises transfecting cell with a nucleic acid construct encoding a NELL1 peptide, and applying the cells in proximity to the region where bone formation is desired

101. The method of claim 96 wherein the nucleic acid construct encoding a NELL1 peptide comprises at least one selected from the group: SEQ ID NO:1, SEQ
ID NO: 3, or SEQ ID NO:5.

102. The method of claim 96 wherein the NELL1 peptide is selected from the group comprising: SEQ ID NO:2, SEQ ID NO: 4, or SEQ ID NO:6.

103. The method of claim 96, wherein the agent is selected from the group comprising: TGF-.beta., BMP2, BMP4, BMP7, bFGF, collagen, bone, matrix, ligament matrix, tendon matrix, osteogenic cells or osteoblastic cells.

104. The method of claim 96 wherein inducing the formation of intramembraneous bone is evaluated by CT, gene expression, histology, mechanical testing.

105. A method of increasing endochondral bone formation comprising:
increasing the concentration of a NELL1 gene product in a region where bone formation and or healing and or regeneration is desired;
optionally applying an agent to the region where bone formation is desired; and at least inducing hypertrophy of chondroblast in the region where bone formation is desired.

106. The method of claim 105, wherein bone formation includes bone healing or bone regeneration.

107. The method of claim 105, wherein the increasing the concentration of a NELL1 gene product comprises applying a NELL1 peptide to the region where bone formation is desired.

108. The method of claim 105 wherein the NELL1 peptide is selected from the group comprising: SEQ ID NO:2, SEQ ID NO: 4, or SEQ ID NO:6.

109. The method of claim 105, wherein the increasing the concentration of a NELL1 gene product comprises inducing the expression of an endogenous NELL1 gene.

110. The method of claim 105, wherein the increasing the concentration of a NELL1 gene product comprises transfecting cell with a nucleic acid construct encoding a NELL1 peptide, and applying the cells in proximity to the region where bone formation is desired

111. The method of claim 110 wherein the nucleic acid construct encoding a NELL1 peptide comprises at least one selected from the group: SEQ ID NO:1, SEQ ID NO: 3, or SEQ ID NO:5.

112. The method of claim 110 wherein the NELL1 peptide is selected from the group comprising: SEQ ID NO:2, SEQ ID NO: 4, or SEQ ID NO:6.

113. The method of claim 105, wherein the agent is selected from the group comprising: TGF-.beta., BMP2, BMP4, BMP7, bFGF, collagen, bone, matrix, ligament matrix, tendon matrix, osteogenic cells or osteoblastic cells.

114. The method of claim 105 wherein inducing the chondroblast hypertrophy causes an increase in apoptotic chondroblasts

115. A method of increasing bone formation in proximity to a vertebra, comprising:
increasing the concentration of a NELL1 gene product proximate to a vertebra where spinal fusion is desired;

optionally applying an additional active agent proximate to a vertebra where spinal fusion is desired; and inducing chondroblast hypertrophy or osteoblast differentiation in the region where spinal fusion is desired.

116. The method of claim 115, further including increasing osteoblastic differentiation.

117. The method of claim 115, further including increasing osteoblastic mineralization.

118. The method of claim 115, further including inducing membraneous bone formation.

119. The method of claim 115, wherein the increasing the concentration of a NELL1 gene product comprises applying a NELL1 peptide to the region where spinal fusion is desired.

120. The method of claim 115 wherein the NELL1 peptide is selected from the group comprising: SEQ ID NO:2, SEQ ID NO: 4, or SEQ ID NO:6.

121. The method of claim 115, wherein the increasing the concentration of a NELL1 gene product comprises inducing the expression of an endogenous NELL1 gene.

122. The method of claim 115, wherein the increasing the concentration of a NELL1 gene product comprises transfecting cell with a nucleic acid construct encoding a NELL1 peptide, and applying the cells in proximity to the region where spinal fusion is desired

123. The method of claim 122 wherein the nucleic acid construct encoding a NELL1 peptide comprises at least one selected from the group: SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.

124. The method of claim 122 wherein the NELL1 peptide is selected from the group comprising: SEQ ID NO:2, SEQ ID NO: 4, or SEQ ID NO:6.

125. The method of claim 115, wherein the agent is selected from the group comprising: TGF-.beta., BMP2, BMP4, BMP7, bFGF, collagen, bone, matrix, ligament matrix, tendon matrix, osteogenic cells or osteoblastic cells.

126. A composition for inducing bone formation comprising:
An effective amount of a first agent to induce bone formation selected from the group comprising: a NELL1 peptide, and an agent that alters expression of NELL1 peptide, or an agent that alters the activity of a NELL1 peptide; and optionally a carrier.

127. The composition of claim 126 wherein the NELL1 peptide is selected from the group comprising: SEQ ID NO:2, SEQ ID NO: 4, or SEQ ID NO:6.

128. The composition of claim 126 wherein the agent is selected from the group comprising: TGF-.beta., BMP2, BMP4, BMP7, bFGF, collagen.

129. The composition of claim 126 wherein the agent is selected from the group comprising: bone matrix, tendon matrix or ligament matrix, osteogenic cells or osteoblastic cells.

130. The composition of claim 126, where the carrier is degradable by enzymatic or hydrolytic mechanisms.

131. The composition of claim 126, where the carrier is comprised a synthetic polymers selected from the group comprising: poly(.alpha.-hydroxy acids) such as poly (L-lactide), poly (D, L-lactide), polyglycolide, poly (lactide-co-glycolide), poly (-caprolactone), poly (trimethylene carbonate), poly (p-dioxanone), poly (-caprolactone-co-glycolide), poly (glycolide-co-trimethylene carbonate) poly (D, L-lactide-co-trimethylene carbonate), polyarylates, polyhydroxybutyrate, polyanhydrides, poly (anhydride-co-imide), propylene-co-fumarates, polylactones, polyesters, polycarbonates, polyanionic polymers, polyanhydrides, polyester-amides, poly(amino-acids), homopolypeptides, poly(phosphazenes), poly (glaxanone), polysaccharides, and poly(orthoesters), polyglactin, polyglactic acid, polyaldonic acid, polyacrylic acids, polyalkanoates.

132. The composition of claim 126, where the carrier is comprised of a cellulosic polymer selected from the group comprising: to alkylcellulose, hydroxyalkylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, carboxymethylcellulose, and their cationic salts.

133. The composition of claim 126, where the carrier is comprised of a synthetic or natural bioceramic selected from the group comprising: calcium carbonate, calcium phosphate, apatite, bioactive glass material, and coral-derived apatite.

134. The composition of claim 126, where the carrier is selected from the group including: collagen, hyaluronan, fibrin, chitosan, alginate, and gelatin.

135. The composition of claim 126, further comprising heparin-binding agents selected from the group comprising dextran sulfate, chondroitin sulfate, heparan sulfate, fucan, alginate.

136. The composition of claim 126, where the carrier is in the form a flowable gel.

137. The composition of claim 126, where the carrier is photopolymerizable.

138. The composition of claim 126, where the carrier is temperature sensitive.

139. The composition of claim 126, where the composition further includes a sequestering agent selected from the group comprising: collagen, gelatin, hyaluronic acid, alginate, poly(ethylene glycol), alkylcellulose (including hydroxyalkylcellulose), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, blood, fibrin, polyoxyethylene oxide, calcium sulfate hemihydrate, apatites, carboxyvinyl polymer, or poly(vinyl alcohol).

140 The composition of claim 126, where the composition further includes a surfactant selected from the group comprising: polysorbate 80, polysorbate 20 or Pluronic F-68.

141. The composition of claim 126, wherein the composition further includes a buffering agent selected from the group comprising: glycine, glutamic acid hydrochloride, sodium chloride, guanidine, heparin, glutamic acid hydrochloride, acetic acid, succinic acid, polysorbate, dextran sulfate, sucrose, and amino acids.

142. A method of treating a patient to increase intramembraneous bone formation, comprising administering NELL1 peptide at a therapeutically effective dose in an effective dosage form at a selected interval to enhance bone formation.

143. The method of claim 142, further comprising administering at least one agent in the region where bone formation is desired selected from the group comprising: TGF-.beta., BMP2, BMP4, BMP7, bFGF, collagen, bone matrix, tendon matrix or ligament matrix.

144. A method of treating a patient to induce bone formation comprising:
harvesting mammalian osteogenic cells;
increasing the concentration of NELL1 peptide in contact with the osteogenic cells;
administering the osteogenic cells to a region where bone formation is desired.

145. The method of claim 144 wherein the increasing the concentration of expression of NELL1 peptide in contact with the osteogenic cells comprises applying a composition including a NELL1 peptide to the osteogenic cells.

146. The method of claim 144 wherein increasing the concentration of expression of NELL1 peptide in contact with the osteogenic cells comprises inducing the endogenous expression of NELL1 peptide.

147. The method of claim 144 wherein increasing the concentration of expression of NELL1 peptide in contact with the osteogenic cells comprises transfecting the cells with a nucleic acid construct expressing NELL1 peptide.

148. The method of claims 145, 146 or 147, wherein the NELL1 peptide is selected from the group comprising: SEQ ID NO:2, SEQ ID NO: 4, or SEQ ID NO:6.

149. The method of claim 145, 146 or 147, further comprising administering at least one agent in the region where bone formation is desired selected from the group comprising: TGF-.beta., BMP2, BMP4, BMP7, bFGF, collagen, bone, matrix, ligament matrix, tendon matrix, osteogenic cells or osteoblastic cells.

150. An implant for use in the human body comprising, a substrate wherein the substrate includes NELL1 in an amount sufficient to induce bone formation proximate to the implant.

151. An implant for use in the human body comprising, a substrate having a surface, wherein at least a portion of the surface includes NELL1 in an amount sufficient to induce bone formation proximate to the implant.

152. An implant for use in the human body comprising: a substrate having a surface, wherein at least a portion of the surface includes osteogenic cells, and the substrate comprises NELL1 in an amount sufficient to induce bone formation.

153. The implant of claim 145, 146 or 147, wherein the substrate is formed into the shape of a mesh, pin, screw, plate, or prosthetic joint.

154. The implant of 145, 146 or 147, wherein the substrate is resorbable.

155. The implant of claim 145, 146 or 147, wherein the substrate comprises collagen.